Molecular Cancer Research - Atrophied Thymus, a Tumor … · The thymus was isolat-ed from cancer cell–inoculated mice and cut into tissue blocks. The tissue blocks were subcutaneously

Cell Death and Survival

Atrophied Thymus, a Tumor Reservoir forHarboring Melanoma CellsOlga Sizova1,2, Denis Kuriatnikov2, Ying Liu2, and Dong-Ming Su1,2

Abstract

Tumor metastatic relapse is the primary cause for cancer-associated mortality. Metastatic relapse is believed to arisefromquantities of tumor cells that are belowdetectable thresh-olds, which are able to resist radio/chemotherapy by obtaininga dormant state and hiding in certain organs, i.e., tumorreservoirs. The thymus, a central T-cell immune organ, hasbeen suggested to be a premetastatic tumor reservoir for B-lymphoma cells. However, it remains unknown whether thethymus is able to harbor nonlymphoid solid tumor cells, andwhether chemotherapy can thoroughly eliminate cancer cellsin the thymus. If chemotherapy is not able to eliminate thesecells in the thymus, then what processes allow for this?Melanoma cell–inoculated and genotoxic doxorubicin-treatedmousemodel systemswere used to determine that the thymus,

particularly the atrophied thymus, was able to harbor bloodstream–circulating melanoma cells. In addition, a chemother-apy-induced DNA-damage response triggered p53 activationin nonmalignant thymic cells, which in turn resulted inthymocyte death and thymic epithelial cell senescence todevelop an inflammatory thymic microenvironment. Thisinflammatory condition induced thymic-harbored minimaltumor cells to acquire a chemoresistant state.

Implications: Here, the thymus serves as a premetastaticreservoir for nonlymphoid solid tumor cells during chemo-therapy, which could be a novel target of minimal residualdisease in antitumor therapy, thus preventing tumor meta-static relapse. Mol Cancer Res; 16(11); 1652–64. �2018 AACR.

IntroductionTumor metastatic relapse at distant organs several years after

removal of the primary tumor and adjuvant chemotherapyposes a clinical challenge. Metastatic relapse results in a poorprognosis and is responsible for the majority of cancer-associ-ated mortality (1, 2). There is a period between primary cureand metastatic relapse, which may be defined as a remissionperiod, when neither symptoms nor cancer cells are detected. Itremains unclear where the cancer cells hide and in whatphysical state they are, as well as why adjuvant chemotherapyis unable to thoroughly eradicate these undetectable tumorcells during the remission period. Emerging evidence hasrevealed that a few cancer cells still survive in certain organsof the body during the remission period. The period of timeduring which these small numbers of cancer cells in the patientsurvive during chemo/radiotherapy is defined as minimalresidual disease (MRD), while these cancer cell–harboringorgans may be defined as premetastatic niches/reservoirs. Bonemarrow (BM) has been determined to be a premetastatic

reservoir for disseminating malignant cells (3–7). The perivas-cular space of blood vessels in the lungs and liver has also beenidentified as these kinds of cancer niches/organs (8, 9). Recent-ly, the thymus has also been identified as a tumor reservoir forlymphoid cancer cells (lymphomas; refs. 10, 11). We askwhether the thymus, the largest T-cell lymphoid organ in thebody, can play a role as a premetastatic reservoir for nonlym-phoid solid tumor cells during chemo/radiotherapy, and if so,why and how the thymus induces its harbored tumor cells toresist chemo/radiotherapy.

The thymus is a primary lymphoid organ responsible forgenerating functional na€�ve T cells and establishing self-tolerance.It undergoes a progressive and age-related involution/atrophy,attributed to the deterioration of the thymic microenvironment(12), which is composed of an integrated three-dimensionalmeshwork of thymic epithelial cells (TEC) and thymocytes.Previously, the thymus was paid scant attention as a cancerpremetastatic reservoir. This may be due to the use of immuno-deficient athymic animal models, such as nude mice, in mostcancer studies. These models with a primary immunodeficiencycannot mimic the natural processes of tumor development andimmune suppression bona fide. Furthermore, the thymus is verysensitive to any physical and chemical assault, particularly che-motherapeutic toxins and radiation, which contribute to thymicatrophy and induce increased proinflammatory factors, such asIL6, thereby potentially becoming a hospitable environment forharboring tumor cells (10, 11). Solid tumor cells can entercirculation as circulating tumor cells (CTC; refs. 7, 13) anddisseminate to distal organs (9), including the thymus (14).

It is well known that inflammation is a double-edgedsword that is necessary for antitumor responses (15–17), butalso induces tumor drug resistance (18–20). For example, theIL6-rich BM microenvironment facilitates chemoresistance

1Cell Biology, Immunology, and Microbiology Graduate Program, GraduateSchool of Biomedical Sciences, FortWorth, Texas. 2Department ofMicrobiology,Immunology, and Genetics, University of North Texas Health Science Center,Fort Worth, Texas.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

CorrespondingAuthor:Dong-Ming Su, University of North Texas Health Center,3500 Camp Bowie Blvd. RES-202F, Fort Worth, TX 76107. Phone: 817-735-5186;Fax: 817-735-2610; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0308

�2018 American Association for Cancer Research.

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in multiple myeloma by up-regulating various prosurvivalproteins (21); and chemotherapy-induced IL6 secretion is alsoinvolved in the ability of cancer cells to acquire stem-likecharacteristics, i.e., generating cancer stem cells (22, 23) thatpossess innate resistance mechanisms to chemo/radiotherapy(24, 25). The stemness-associated features are potentiallydue to activation of antiapoptotic features (26) and inductionof the cancer cell–intrinsic quiescent state of G0 to G1 arrest(27). The chemotherapy-resistant feature is attributed tomicroenvironment-induced tumor cell changes in gene expres-sion profiles. For example, some genes are turned on, suchas MAPK p38, while others are turned off, such as MAPKERK. A high ratio of p38/ERK (activation of p38 and inhibitionof ERK) induces tumor growth arrest (dormancy: a risk forcancer recurrence), while a high ERK/p38 ratio favors tumorregrowth (28–30).

In this report, we first focused on determining whether thethymus is able to retain circulating solid nonlymphoid mela-noma cancer cells. We then addressed how the atrophiedthymic microenvironment becomes a suitable tumor reservoirduring chemotherapy. Finally, we demonstrated how thymic-harbored melanoma cells acquired chemoresistance by expo-sure to the inflammatory factor–rich thymic microenviron-ment. We determined that when the thymus (lymphoid organ)is in an inflammatory condition, it is able to harbor circulatingnonlymphoid solid cancer cells. This inflammatory microen-vironment results from chemotherapy-induced DNA-damageresponses (DDR), thereby triggering p53 activation in thymiccells. This, sequentially, results in thymocyte death and TECsenescence. Thus, an inflammatory condition is developed tofacilitate thymic-harbored minimal tumor cells to acquire anantiapoptotic predominant chemoresistant feature. Together,our results have identified a novel premetastatic cancer reser-voir: the thymus. We bring this novel target into the focus forantitumor therapy of nonlymphoid solid tumor cells to combatpotential metastatic relapse.

Materials and MethodsMice and animal care

All animal experiments were performed in compliance withprotocols approved by the Institutional Animal Care and UseCommittee of the University of North Texas Health ScienceCenter, in accordance with guidelines of the NIH. C57BL/6wild-type (WT) young (1–3 months old) and aged (�18 monthsold, purchased from the rodent colonies of National Institute onAging) were used. Two gene manipulated mouse colonies werealso used: (i) FoxN1 conditional knockout (termed FC) mice.Once the FoxN1-floxed gene is deleted via CreERT activationby induction with three intraperitoneal (i.p.) injections oftamoxifen (TM), the thymus becomes atrophied (details in ourpreviously published paper, ref. 31); (ii) immunodeficient NSG(NOD. Cg-Prkdc-scid, and il-2rg�/�) mice.

Tumor cell lines and GFP transduction, the generation ofcirculating tumor cell condition in mice, and subcutaneousimplantation of tumor-bearing tissues

Mouse melanoma B16F1 cells (ATCC, CRL-6323, simplytermed "B16") were transducedwith enhanced greenfluorescenceprotein (eGFP) lentivirus particles containing neomycin resis-tance gene. On 80% confluent B16 cells, cultured in a 24-well

plate with 1mL of complete DMEMmedium containing 5 mg/mLof polybrene, 12 mL of eGFP lentiviral particles (>1 � 108,from GeneCopoeia, Cat#: LPP-EGFP-LV151-100-C) were add-ed. Three days later, the GFPþ cells were visualized by fluore-scence microscopy, and GFPhi cells were sorted via InfluxCell Sorter (BD Biosciences). GFP-expressing B16 cells werecultured in complete growth medium DMEM, supplementedwith 500 mg/mL of G418 (for neomycin maintenance of thecells). When the confluence was about 80% to 90%, the cellswere dissociated for single-cell suspension with 0.25% Trypsin-1mmol/L EDTA solution followed by washing with 1� PBS twice.This single-cell suspension was used for intravenous (i.v.) injec-tion (1� 106 cells/mouse) through retro orbital route to mimic acirculating tumor cell condition in mice. The thymus was isolat-ed from cancer cell–inoculated mice and cut into tissue blocks.The tissue blocks were subcutaneously transplanted into immu-nodeficient NSG mice under the dorsal skin. Three to 4 weeksafter the transplantation, the mice were sacrificed, and tumor wasvisualized under the skin.

Tumor recurrence in vitro assayWT mice received i.v. inoculation with 1 � 106 B16-GFP

melanoma cells. Three days later, the mice were treated withdoxorubicin (Doxo) or PBS. The thymus, lymph nodes (LN),and lungs were adjusted to similar weight and individuallycultured in a plate, and 10 to 14 days later, the GFPþ cells werevisualized and semiquantitatively measured with ImageJsoftware.

Flow cytometryTo analyze the percentage of cancer cells in the thymus and

LNs, single-cell suspensions were prepared using enzymaticdigestion. Freshly isolated tissues were torn apart and digestedat 37�C in DNase-I/Collagenase V solution, as previouslydescribed (32). The single-cell suspensions were then stainedwith specific antibodies on cell surface markers and/or intra-cellular GFP, phosphoralated-p53, or Ki67, and fixed with 2%PFA for 1 hour and permeabilized with 0.1% Triton X-100,as previously reported (32). Fluorochrome-conjugated anti-bodies against CD45 (30-F11), MHC-II (M5/114.15.2),EpCAM (G8.8) were purchased from BioLegend. The anti-GFP(BioLegend, Cat #338002), antiphosphorylated p53 antibody(Ser15, Cell Signaling Technology, Cat #9284) with secondaryAlexa Fluor 488–conjugated donkey anti-Rabbit IgG antibody(Invitrogen, Cat #Z-25302) were used. Flow cytometry wasperformed using an LSRII flow cytometer (BD Biosciences) anddata were analyzed using FlowJo software.

ELISA assay for inflammatory cytokines in thymic tissuesThymic tissues were freshly isolated and homogenized in

RIPA buffer (Sigma, Cat #R0278). Total protein concentrationswere measured using the BCA Protein Assay Kit from ThermoFisher (Cat #23227). IL6, IL1b, and TNFa were quantified byELISA (from BioLegend, Cat #431305, Cat #432605, and Cat#430905), following the manufacturer's instructions. Standardcurves for IL6, IL1b, and TNFa were generated with a range of0–500 pg/mL. Samples were run in duplicate and the datarepresent the mean of multiple animals (indicated in thefigures). The substrate was TMB (3,30,5,50-tetramethylbenzi-dine) and the absorbance was measured at 450 nm with theBioTek ELx800 ELISA reader.

Atrophied Thymus as a Tumor Reservoir

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Annexin-V–based and caspase-3–based apoptotic assays inthymocytes, TECs, and/or cultured B16 tumor cells

Thymocytes and TECs were freshly and enzymatically isolatedfromDoxo- or PBS-treated youngWTmice. Cells were stained forsurface markers, washed, and incubated in APC–Annexin-V anti-body (BioLegend, Cat #640920) at a 1:20 dilution with Annexin-Vbuffer (10mmol/LHepes adjusted to pH7.4, 140mmol/LNaCl,and 2.5 mmol/L CaCl2) for 15 minutes at room temperature,followed by flow-cytometric analysis. In addition, we did caspase-3–based apoptotic assaywith flow cytometry to confirmAnnexin-V results, in which cleaved caspase-3 (Asp175) monoclonalantibody (Cell Signaling Technology, Cat #9579T) and secondantibody (Alexa Fluor 488–conjugated anti-rabbit IgG, Zenon,Cat #Z-25302) were used for intracellular staining. Apoptoticpositive control cells were prepared by incubating cells at 55�Cfor 20 minutes to induce cell death before staining. Details weredescribed in our recent publication (33).

Cryosections for immunofluorescence (IF) or SA-b-Gal(senescence) staining

Cryosections (5–6 mm thick) were stained as described pre-viously (32). The tissue was fixed with acetone, and then stainedwith various antibodies. The primary antibodies used wereanti-K8 (Troma-1 supernatant), anti-GFP (B2; Santa CruzBiotechnology, Cat #sc-9996), anti-p21 (C-19; Santa CruzBiotechnology, Cat #sc-397), anti-p16 (F-12; Santa Cruz, Cat#sc-1661), or anti-TAp63a (N-16; Santa Cruz Biotechnology,Cat #sc-8609). Based on primary antibody species, the second-ary antibodies used were Cy3-conjugated or Alexa Fluor 488–conjugated donkey anti-rabbit or anti-rat IgG (Jackson Immu-noResearch Lab) or (Invitrogen, Cat #Z-25302). For senescence-associated b-galactosidase (SA-b-Gal) staining, cryosections ofyoung and aged, as well as FCmouse thymus tissues (8 mm thick),were analyzed for SA-b-gal activity using a senescence b-Galacto-sidase staining kit according to the manufacturer's protocol(Cell Signaling Technology, Cat #9860) and counterstained withnuclear fast red (RICCA Chemical #R5463200) solution.

Western blot analysis of p53 expressionThe whole thymus was subjected to homogenization and

protein extraction in RIPA lysis buffer (Sigma, Cat #R0278),containing 1� protease inhibitor cocktail (Sigma, Cat #P8340)and 1� phosphatase inhibitor cocktail (Sigma, Cat #P0044).Protein, �30 mg/lane, was loaded under reducing conditions fora directWestern blot assaywith antiphosphorylated p53 antibody(Ser15; Cell Signaling Technology, Cat#9284) and anti-total p53antibody (Santa Cruz Biotechnology, Cat#sc-6243), respectively.Housekeeper GAPDH or b-actin was used as an internal loadingcontrol. Positive protein bands were visualized through Super-Signal West Femto Maximum Sensitivity Substrate (Thermo Sci-entific, Cat #34095) and scanned by a C-Digit Scanner (LI-COR).

Transwell cell culture and in vitro cell death and proliferationanalysis

B16 cancer cells with 10% to 30% confluence were treated with3 mmol/L Doxo for 8 hours. Thymuses of young C57/BL6 miceinjected with PBS or Doxo (2 times for 10 mg/kg mouse weight)were placed on themembrane of the transwell and cocultured for3 to 5 days. Then, cancer cells were detached from the bottom ofthe plate using a nonenzymatic dissociation solution (Sigma, Cat#c5789) andused for furtherflow-cytometric analysis of Annexin-

V–based apoptosis and/or Ki67 (BioLegend, Cat #652404) pro-liferation assay, and/or evaluation of dormancy phenotype byflow-cytometric analysis using p-p38/p-ERK ratio (Cell SignalingTechnology, Cat #4551S and #4375S).

Statistical analysisFor evaluation of group differences, an unpaired two-tailed

Student t test was used assuming equal variance. Differenceswere considered statistically significant at values of �, P < 0.05and ��, P < 0.01.

ResultsThe thymus, particularly the atrophied thymus, can harbormelanoma (nonlymphoid solid tumor) cells with a regrowthcapacity

In order to determine whether the thymus, a lymphoid organ,was able to harbor circulating nonlymphoid solid tumor cells, wei.v. injected mouse melanoma cells (B16 cells transduced withenhanced green fluorescent protein, eGFP) into immunocompe-tent WT mice, which arbitrarily mimics the situation in whichcirculating solid tumor cells (13) disseminate to distal organs (9).Seven to 10days after the inoculation,wewere able to detectGFPþ

melanoma cell clusters in the normal thymus (Fig. 1A, top).Although thymic tissues usually show autofluorescence signals,we can easily distinguish between this and the PBS-inoculatedcontrol mice (PBS-Ctr; Fig. 1A, bottom) and the B16-GFPþ cell–inoculated mice (Fig. 1A, top). To confirm whether these GFPþ

cell clusters were the inoculated GFPþ melanoma cells, we iso-lated the thymuses from both B16-GFP and PBS-Ctr group miceand transplanted these thymuses (either cancer cell–bearing orcontrol) into immunocompromised NSG (NOD. Cg-Prkdc-scid,and il-2rg�/�) mice subcutaneously (procedure shown in Fig. 1B,left). Around 4 weeks after the transplantation, we found thattumors developed under the skin of the NSG mice transplantedwith the thymus frommelanoma-inoculatedmice (Fig. 1B, right),but we did not find any developed tumor until 8 weeks after thegraft in the NSG mice transplanted with the thymus of PBS-Ctrmice (image not shown). This evidence suggests that the body'slargest T lymphoid organ, the thymus, is able to harbor non-lymphoid solid tumor cells, in addition to lymphoid fluid cancercells (10, 11), with a regrowth capacity.

The thymus undergoes atrophy generating a proinflamma-tory microenvironment once it receives any physical and chem-ical assaults, including chemotherapy. We wanted to determinewhether the atrophied thymus was more hospitable for har-boring nonlymphoid solid tumor cells. We compared two typesof atrophied thymuses. One is the FoxN1 conditional geneknockout (FC) thymus in adult young mice, which has aninducible defect in TEC homeostasis (31). The other is naturallyaged thymus from over 18-month-old mice. With the samestrategy as in Fig. 1A, we found that both types of atrophiedthymuses were able to harbor higher proportion of melanomacells than the normal young thymus did with analyses of bothflow cytometry (Fig. 1C) and immunofluorescence staining(Fig. 1D). This phenotype can also be observed in the atrophiedthymus of FC mice with i.v. inoculation of lymphoid lympho-ma cells (Supplementary Fig. S1). These results indicate that themicroenvironment in the atrophied thymus is a particularlysuitable environment for harboring both lymphoid and non-lymphoid solid cancer (melanoma) cells.

Sizova et al.

Mol Cancer Res; 16(11) November 2018 Molecular Cancer Research1654




Chemotherapeutic drug induces thymic atrophy to generate aninflammatory microenvironment, attributed to a combinationof thymocyte death and TEC senescence

The capacity of the atrophied thymus to harbor cancercells with the ability to regrow is related to the thymic inflam-matory microenvironment (10, 11), which results from chemo/radiotherapy. Chemotherapeutic drugs can induce cell stress–associated DDR, resulting in cell death or/and senescence

(34, 35). In the thymus, chemotherapeutic drugs impact on notonly malignant tumor cells, but also potentially nonmalignantthymic cells, which consist of hematopoietic thymocytes andnonhematopoietic TECs (36). To verify whether chemotherapycould induce thymic atrophy, and how inflammatory thymicmicroenvironment was generated, we treated mice with a com-monly used antitumor genotoxic drugDoxo toobserve changes inthe young healthymurine thymus. As shown in Fig. 2A,mice were

Figure 1.

The thymus, particularly the atrophiedthymus, is able to harbor melanoma(nonlymphoid tumor) cells with acapacity for regrowth. Mice were i.v.inoculated with mouse GFP-transduced B16F1 (termed B16-GFP)melanoma cells (1� 106 per mouse) orPBS for control (PBS-Ctr), 1 week afterthe inoculation the thymuses wereexamined. A, Thymic cryosection(from young WT mice) fluorescencestaining shows one representativeresult of GFPþ cells in the B16-GFP-inoculated thymus (top) but not in thePBS-Ctr thymus. The data arerepresentative of 5 biologicalreplicates in each group withessentially identical results. B, Left,Experimental schema of thymictissues from WT mice in A to NSGmice. Right, Tumor regrowth from thethymic tissue of B16-GFP–inoculatedWT mice under the NSG mouse skin.However, no tumor growth wasobserved from the thymic tissues ofPBS-Ctr mice (image is not shown).The image is a representative resultfrom at least three independentexperiments (n ¼ animal numbers). C,Left, Flow-cytometric plots showgates of thymic-harbored B16-GFPmelanoma cells in the thymuses ofthree types ofmice (from left to right):Young WT, FoxN1 conditional geneknockout (FC), andWT naturally agedmice. Right, A summarized result of %GFPþ cancer cells in the thymusesshown in a bar graph. A Student t testwas used to determine statisticalsignificance between groups, and dataare expressed as mean � SEM. D,Freshly isolated thymic cryosectionsfrom 3 groups of mice (same as C)were stained with fluorescenceantibodies and visualized. One of therepresentative results shows GFPþ

cells in the B16-GFP–inoculatedthymuses (top), but not in the PBS-Ctrthymuses (bottom). Data arerepresentative of 3 biologicalreplicates in each group withessentially identical results.






intraperitoneally (i.p.) injected with Doxo at 8 to 10 mg/kg bodyweight or PBS for 3 consecutive days (once a day), and 3 days afterthe last injection, their thymuses were observed. The thymuses inDoxo-treated mice were dramatically reduced in size, weight, andtotal thymocyte numbers (Fig. 2A). We also found that anincreased proinflammatory condition (after 4–5 Doxo injectionsat 10 mg/kg) was induced by the chemotherapeutic drug in theatrophied thymuses, exhibited by increased IL6, IL1b, and TNFacompared with the normal thymuses (Fig. 2B). The results con-firm that chemotherapy indeed induces thymic atrophy andestablishes a proinflammatory thymic microenvironment.

We hypothesized that the Doxo-induced inflammatorythymic microenvironment should be attributed to thymic celldeath and/or cellular senescence. Then, we checked theseparameters in the thymuses of Doxo-treated and PBS-treated(PBS-Ctr) young WT mice (Fig. 3). Using Annexin-V–basedapoptotic assay, we found that apoptosis in thymocytes fromDoxo-treated mice was increased, while apoptosis in TECs wasnot, compared with their PBS-Ctr counterparts (Fig. 3A, left).In order to confirm this result, we checked cleaved caspase-3in these cells. Activation of caspase-3 plays a central role in theexecution-phase of cell apoptosis induced by either intrinsic(via p53) or extrinsic (via TNF receptor) apoptotic pathways(37). The results were consistent with our Annexin-V–basedapoptosis assay (Fig. 3A, right). We further asked what hap-pened in TECs after Doxo-treatment. We examined a senescentphenotype in these TECs, with a positive control group of8-month-old FC thymus that has increased TEC senescence(32). We noted that two senescence-associated molecules, p21(CDKN1A) and p16INK4A, were increased in the thymuses of

Doxo-treated mice (Fig. 3B). To confirm this, we performedSA-b-gal staining (a senescence marker) of thymic cryosectionsand observed that a senescent phenotype had developed inthe thymus of Doxo-treated mice (Fig. 3C). To verify thesenescent phenotype was in the TEC population, we stainedthe thymic cryosections with the TAp63 marker, which isrelated to senescence and is expressed only in TECs (32).Expression of TAp63 was indeed increased in the thymusesof Doxo-treated mice (Fig. 3D). In addition, we costained thethymic cryosections with TAp63 and p21 and confirmed analmost complete colocalization of TAp63 (increased expres-sion in senescent TECs) versus p21 (senescence-associatedmolecule; Supplementary Fig. S3), which we reported in ourprevious publication (32). This further proves that senescenceoccurs in TECs during chemotherapy. Therefore, the findingthat Doxo-treatment resulted DDR-induced changes in thethymus have a feature of apoptosis mainly in thymocytes andsenescence mainly in TECs has been confirmed.

We know that nonmalignant thymic cells mainly include twopopulations: hematopoietic thymocytes and nonhematopoieticTECs. Our results reveal that the chemotherapeutic drug affectsboth thymic cell populations, and the proinflammatory conditionis synergistically composed of both increased cell death in thy-mocytes accompanied by increased cellular senescence in TECs.Increased cellular senescence may be further involved in thesenescence-associated secretory phenotype (SASP; refs. 38, 39)to participate and/or enhance the development of a thymicproinflammatory condition, and senescence-associated IL6 andIL8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities (40).

Figure 2.

Chemotherapy induces thymic atrophy to generate an inflammatory thymic microenvironment. Young WT mice were intraperitoneally (i.p.) injected witheither genotoxic drug Doxo at 10 mg/kg body weight or PBS once a day for 4–5 times, with an interval resting day in between. Three to 5 days afterthe last injection, the thymuses were collected for analysis. A, Left, A representative image of the thymuses shows the thymic atrophy in the Doxo-treatedgroup, but not in the PBS-Ctr group; middle, a summary of ratios of thymus/body weight (BW) in mg; right, a summary of total thymocyte numbersin the two groups. B, A summarized result of three types of proinflammatory cytokines in the thymuses of the two groups. Left, Concentration ofcytokine product in pg/mg of thymic protein; right, relative production in fold changes (baseline set as the average of cytokine concentrations in PBS-treatedgroup as 1). A Student t test was used to determine statistical significance between two groups. All data are expressed as mean � SEM. The P values areshown in each panel, and each symbol represents an individual animal sample.

Sizova et al.





Figure 3.

Chemotherapy (Doxo-treatment) induces increased apoptosis in thymocytes and senescence response in TECs. Mice were treated with 3 consecutivei.p. injection Doxo (10 mg/kg) or PBS once a day. Three days after the last injection, the thymuses were collected for analysis. A, A summary ofapoptotic analysis of thymocytes (gated on the CD45þEpCam�neg population) and TECs, gated on the CD45�negEpCamþ population) with Annexin-Vassay (left) and cleaved caspase-3 assay (right), respectively. Relative fold changes were based on setting an average of % Annexin-Vþ cells or %cleaved caspase-3þ cells in PBS-Ctr thymocytes and TECs as 1, respectively. All data are expressed as mean � SEM. The P values are shownbetween two compared groups, each symbol represents an individual animal sample. B, Representative thymic cryosections with immunofluorescencestaining show the images of keratin-8 (counterstaining) vs. p21 in the top row and p16INK4A in bottom row from the thymuses of three types of mice.Eight-month-old FC thymus served as aged control because it is similar to the 18-month-old naturally aged thymus (33). C, Representative images ofthymic cryosections with SA-b-gal staining (blue clusters) vs. nuclear fast red counterstaining from three types of thymuses as B. D, Representativefluorescence images of thymic cryosections with TAp63 (a senescent TEC marker; ref. 32) staining (red clusters) vs. keratin-8 counterstaining fromthree types of thymuses as B. Arrows in B–D show typical positive cell clusters. Image data are representative of 3 to 4 animals in each group withessentially identical results. The rightmost column is semiquantitative data obtained via ImageJ software, and each symbol represents the ratio of %positive area per scope (9–14 tissue scopes were recorded for each animal) of the tissues from 3–4 animals in each group.






Changes in thymic microenvironment are potentiallycorrelated with DDR-triggered activation of p53 gene

To determine the potential mechanism of drug-inducedthymocyte apoptosis and TEC senescence, we hypothesized thatp53 gene would be involved in these processes based on itsfunction in promoting both cellular apoptosis and senescence(41, 42), as well as the fact that activation of p53 is commonlytriggered by the cell stress–associated DDR (34, 43). To verify itscorrelation to chemotherapy-induced thymic cellular apoptosisand senescence here, we first measured phosphorylated (activat-ed) p53 (P-p53) and total p53 in the thymus from Doxo-treatedmice with Western blot assay and found both were increasedcompared with those from PBS-Ctr mice (Fig. 4A). Then,

we determined which thymic cell populations had increasedP-p53 with flow-cytometric analysis. We found that Doxo-treatment increased the percentage of P-p53þ cells in both hem-atopoietic thymocytes and nonhematopoietic TECs (Fig. 4Band C). However, it exhibited uniform increase in TECs (smallstandard deviation), but a heterogeneous increase in thymo-cytes (a large variability) of the Doxo-treated mice (indicatedby a broken-line circle in Fig. 4C). Our results demonstratethat both increased cell death and development of senescencein the thymus during chemotherapy are associated with acti-vation of p53 in nonmalignant thymic cells, which is triggeredby antitumor drug-induced DDR stress.

Inflammatory thymic microenvironment ofchemotherapeutic drug-treated mice confers tumorcells toward a chemoresistant phenotype

Based on evidence that the inflamed atrophied thymus isable to harbor nonlymphoid solid cancer cells during chemo-therapy, we wanted to know why the chemotherapy is not ableto completely eradicate the thymic-harbored cancer cells andwhether these thymic-harbored tumor cells are conferred achemoresistant phenotype by this inflammatory condition. Weinoculated B16-GFP melanoma cells (1 � 106 per injection) toyoung WT mice, and 3 days after the inoculation, we treatedthese tumor-bearing mice with Doxo for 3 days (8–10 mg/kg,once a day for 3 consecutive days). Three days after the last drugtreatment, we compared the tumor cells in the thymus and LNsand found that the ratio of percentage of melanoma cells inthymus versus lymph nodes was increased (Fig. 5A), implyingthat chemotherapy killed cancer cells more efficiently in theLNs than it did in the thymus. In other words, cancer cellsharbored in the thymus were more resistant to chemotherapy.Because Doxo treatment usually results in autofluorescentinterference during flow-cytometric assay in Peridinin Chloro-phyll Protein Complex (PerCP) and Alexa Fluor 488 (AF488)channels (Supplementary Fig. S2), we made an effort to avoidusing PerCP-conjugated antibodies, and strictly set up a cutofffor the positive population for the GFP channel. In addition,we directly visualized the inoculated GFPþ melanoma cell clus-ters with immunofluorescent staining of thymic cryosectionsfrom mice with the two treatments (Doxo or PBS) and con-firmed that a proportion of these clusters was increased in thethymus from Doxo-treated mice (Fig. 5B). In order to furtherconfirm that chemotherapy has little effect on the thymic-harbored cancer cells, we compared the absolute melanomacell numbers using a flow-cytometric approach (Fig. 5A, left)after inoculation and treatment with/without Doxo. Weobserved that the absolute cell numbers of the GFPþmelanomacells in the thymuses from Doxo-treated mice were not reduced(Fig. 5C). The results indicate that the chemotherapeutic drugtreatment induces thymic atrophy and dramatically reducedthymic mass, but this did not significantly affect numbers ofthymic-harbored cancer cells.

We next attempted to investigate whether the chemothera-peutic drug was able to kill melanoma cells equally in thethymus, LNs, and lungs. The lungs are the most suitablemetastatic site for melanoma (44). We inoculated B16 mela-noma cells into young WT mice and then treated the mice withDoxo or PBS twice (10 mg/kg, once a day for 2 consecutivedays). Three days after the last drug treatment, we isolated thethymus, LNs, and lungs, and cultured these three tissues

Figure 4.

Chemotherapy (Doxo-treatment) induces activation of p53 in the thymus.With the same treatment as those in Fig. 3. A, Western blot analysis ofphosphorylated p53 (P-p53; left) and total p53 (right) in the thymuses ofthe two groups. B, Flow-cytometric gate strategy of thymocyte(CD45þMHC-II�neg) and TECs (CD45�negMHC-IIþ), as well as P-p53þ cellpopulations. C, A summary of percentages of P-p53þ cells in TECs andthymocytes of PBS-Ctr and Doxo-treated groups, respectively. A Studentt test was used to determine statistical significance between groups. Alldata are expressed as mean � SEM. The P values are shown in each panel,and each symbol or "n" represents an animal or animal numbers.

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adjusted to similar weight in separate plate wells for about 2weeks. On the final day of the culture, we visualized the cancercell regrowth via GFPþ clusters under the microscope (Fig. 6A)and semiquantitatively measured these green cellular clusterswith ImageJ software (Fig. 6B). We found that the inoculatedcancer cells in both the thymic and lung tissues from mice withchemotherapy could regrow, but regrowth of cells from thethymic tissues was greater than that from the lung tissues. Weset up the baseline for our calculations as follows: the percent-age of GFPþ cell cluster area in PBS-treated mice as 100%(because the cancer cells in cancer cell–bearing tissues withoutdrug treatment should fully regrow). The percentage of GFPþ

cancer cell cluster area in Doxo-treated mice was evaluatedin comparison with the 100% regrowth baseline in the cor-responding tissue with PBS-treatment using ImageJ software

(Fig. 6B). In addition, we seldom found any cancer cell re-growth from the LN tissues of Doxo-treated mice.

Taken together, our results suggest that the chemotherapeu-tic drug is not able to equally kill cancer cells in the thymus,LNs, and lung. Particularly, the thymus from Doxo-treatedmice harbored more cancer cells. This is due to an inflamma-tory environment, which may direct the harbored cancercells toward a chemoresistant feature. Although these cancercells cannot be completely eradicated in the thymus, they areunlikely to develop a tumor in the thymus, because we neverobserved thymoma development in these mice. Therefore,the atrophied thymus is only a potential tumor reservoir. Thecancer cells in the lungs were also not completely eradicated,in accordance with previous findings identifying the lungs asa known tumor reservoir (9).

Figure 5.

Chemotherapy induces achemoresistant phenotype in thymic-harbored melanoma cells. With thesame melanoma (B16-GFP)inoculation as in Fig. 1, and then thesame Doxo-treatment as thosein Fig. 3. Three to 5 days after the lastdrug injection, the thymuses andinguinal and mesenteric LNs wereisolated for analysis. A, Left, Aflow-cytometric gate strategy showsmelanoma cells (GFPþCD45�neg

population) in the thymuses (top) andLNs (bottom); right, summarizedratios of % melanoma cells in thethymus-to-LN of PBS-Ctr andDoxo-treated groups. B, Left,Immunofluorescence staining imagesof GFPþ melanoma cell clusters(Green) in Keratin-8 TEC background(Red) of three differently treatedmouse thymuses. The bar in the imageis 100 mm. Right, A summary of GFPþ

melanoma cell clusters in thethymuses of three differently treatedmice. The GFPþ cell cluster imageswere semiquantitatively analyzedusing ImageJ software. C,Summarized flow-cytometric data forabsolute cancer cell numbers (gatedon GFPþCD45�neg) in the thymusesfrom B16 cell–inoculated and PBS-Ctror Doxo-treated mice. A Student t testwas used to determine statisticalsignificance between groups. All dataare expressed as mean � SEM. Dataare pooled from at least three scopesper independent slide per animalthymus. The P values are shown ineach panel, each symbol or "n"represents an animal or animalnumbers.






Thymus from chemotherapeutic drug-treated mice potentiallymodulates the thymic-harbored cancer cells to acquire anantiapoptotic feature

Because thymic-harbored cancer cells were able to resist che-motherapy, we assumed that the drug-induced inflammatorysoluble factor–rich microenvironment (10) in the thymus mod-ulates the cellular features (proliferation and apoptosis) in thethymic-harbored cancer cells. We designed a novel analysis sys-tem, through an in vivo (by drug-induced changes in the thymus)plus in vitro (by a transwell coculture system, to confer thesechanges to a drug-pretreated cancer cells) system (work schema isshown in Fig. 7A). The results showed that when the atrophiedthymus from Doxo-treated mice was cocultured with Doxo-pre-treated B16 cancer cells in a transwell (Fig. 7A, group #4), thesecancer cells were more resistant to Doxo-induced apoptosis, witha reduced percentage of Annexin-Vþ cells (Fig. 7B: the filled peak,indicated by an arrow in the left histograms panel labeled with"Doxo-treated thymus," and the rightmost filled striped bar in theright).With the same transwell coculture system,we did not find asignificant difference in proliferation (Fig. 7C: an arrow in the leftlabeled with "Doxo-treated thymus," and the rightmost stripedbar on the right). The cellular features of decreased apoptosis with

unchanged proliferation in the cancer cells cocultured with theatrophied thymus from Doxo-treated mice suggests that drug-induced thymic soluble factors, mostly proinflammatory factors,indeed confer antiapoptotic feature to the thymic-harbored can-cer cells.

Furthermore, we wanted to answer why proliferation in thesecancer cells was not changed. We believe that the thymic-harbored cancer cells could obtain intrinsic changes via mod-ulation by the inflammatory (Doxo-treatment) thymus. Weperformed intracellular staining of P-p38 and P-ERK withflow-cytometric analysis, because a high ratio of P-p38/P-ERK(activation of p38 and inhibition of ERK) induces tumorgrowth arrest, i.e., dormancy, while a high P-ERK/P-p38 ratiofavors tumor regrowth/recurrence (28–30). We found that theratio of P-p38/P-ERK was significantly increased in the cancercells under the transwell coculture with the atrophied thymusfrom Doxo-treated mice (Fig. 7D, the rightmost filled stripedbar in right). The results indicate that the atrophied thymusfrom Doxo-treated mice confers Doxo-pretreated cancer cellswith a relative dormant feature.

Taken together, thymic-harbored cancer cells in the inflamma-tory thymus possess the capacity to resist chemotherapy through

Figure 6.

Capacity of cancer cell regrowth fromthe thymus is greater compared withother organs from the mice withchemotherapy. WT young mice wereinoculated with B16-GFP cancer cells,and then treated with Doxo or PBS,same setting as in Fig. 5. Three daysafter the last drug injection, thethymus, LNs, and lungs were freshlyisolated. These three tissues with thesimilar weight were cut into smallpieces and homogenized, then eachorgan was subsequently cultured inseparate platewells for about 2weeks.On the final day of the culture, GFPþ

cell clusters were visualized andsemiquantitatively measured withImageJ software. A, Representativeimages of GFPþ melanoma cells inculture from three types of tissues (thethymus, LNs, and lung) of PBS-Ctr(top) and Doxo-treated (bottom)groups, respectively. B, Summarizedratios of % of GFPþ areas derived fromDoxo-treated mice to the areasderived from PBS-treated mice. Eachsymbol represents ratio of % positivearea per scope of the tissues from atotal of 4 animals in each group.This experiment was repeated at least4 times. All data are expressed asmean � SEM. The P values are shownin each panel.

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Figure 7.

The atrophied thymus from Doxo-treated mice modulates Doxo-pretreated melanoma cells toward exhibiting increased antiapoptosis but unchangedproliferation, and increased ratio of P-p38-to-P-ERK. A, Experimental schema of thymic modulation on B16 melanoma cells. In this system, we i.p. injected youngWT mice with Doxo as we did in Fig. 3. We isolated the thymuses from the Doxo-treated and PBS-Ctr mice, respectively, and cut the thymuses intotissue pieces. We loaded the same weight of thymic tissue blocks on top of transwells to coculture with Doxo-pretreated B16 melanoma cells in monolayerculture on the bottom wells. Three days after the coculture, in which the thymus and monolayer B16 cells were separated by the transwell membrane, theB16 cancer cells were analyzed with flow-cytometric assays. B, Results of Annexin-V–based apoptosis assay. Left, Representative histograms show Annexin-Vþ

B16 melanoma cells with or without modulation by the thymuses from PBS- and Doxo-treated mice, respectively. Right, Summarized results show % ofAnnexin-Vþ PBS/Doxo-pretreated B16 cancer cells in 4 different groups (the bars from left to right match the order of the group #1 to #4 in A). C, Resultsof Ki67-based proliferation assay. Left, Representative histograms show Ki67þ proliferative B16 melanoma cells with or without modulation by thethymuses of PBS- and Doxo-treated mice, respectively. Right, Summarized results show % of Ki67þ PBS/Doxo-pretreated B16 cancer cells in 4 different groups(the bars from left to right match the order of the groups #1 to #4 in A). D, Results of intracellular staining of P-p38 and P-ERK, respectively. Left, Representativehistograms show P-p38þ and P-ERKþ B16 melanoma cells with or without modulation by the thymuses of PBS- and Doxo-treated mice, respectively.Right, Summarized results show ratios of P-p38þ to P-ERKþ PBS/Doxo-pretreated B16 melanoma cells in 4 different groups (the bars from left to right matchthe order of the groups #1 to #4 in A). All data are expressed as mean �SEM. The P values are shown in each panel, and "n" represents animal numbers.






antiapoptosis accompanied by unchanged proliferation, attrib-uted to thymus-modulated intrinsic changes in molecular acti-vation, such as increased P-p38 and decreased P-ERK, whichinduces a feature of drug-resistant dormancy.

DiscussionTo combat tumor metastatic relapse, it is important to identify

premetastatic reservoirs, because they retain MRD, resulting ineventual tumor relapse. Several cancer premetastatic reservoirs,such as BM, which preserve disseminating malignant cells (3–7),and the perivascular space of blood vessels in the lung and liver,which serve as cancer niches (8, 9), have been determined.However, in all likelihood, these are not the only tissues that canserve as tumor reservoirs for harboring MRD, because the chal-lenge of tumormetastatic relapse is still unsolved. The thymus hasbeen determined to be a B-lymphoma premetastatic reservoir inrecent studies (10, 11). We confirmed the role of the thymusserving as a premetastatic reservoir not only for lymphoid cancercells (Supplementary Fig. S1), but also for nonlymphoid mela-noma cells. We also determined how chemotherapy induces thethymus to form a tumor reservoir and how this reservoir canmodulate the thymic-harbored tumor cells to acquire a chemo-resistant feature, the dark side of chemotherapy. When patientswith cancer receive chemotherapy, there is risk to damagehealthy tissues, including creating an atrophied and inflammatorythymus that potentially harbors circulating cancer cells. Theinflammatory thymic microenvironment, in turn, protects andmodulates its harbored cancer cells to enter a chemoresistant statewith intrinsic signaling alteration. This is a risk because thesethymic-harbored dormant cancer cells could eventually develop anew tumor once conditions are suitable for them to disperse todistant organs.

The model we use in Figs 1, 5, and 6 to mimic CTCs wasgenerated through an i.v. injection of B16 tumor cells. Werecognized that this is a relatively artificial means of mimickingspontaneously tumor cell spread. However, by studying thedistribution of CTC via the bloodstream rather than metastasisitself, this model has its advantages (45), including control-lable numbers of cells delivered to each mouse, and compa-rability between each experiment, short waiting time for evi-dence, and easy observation by flow cytometer and fluorescentmicroscopy. This model may not be ideal for studying themechanism of metastasis, but it is suitable for determiningtumor reservoirs, and particularly for preliminary assessmentof how the reservoir microenvironment interacts with its har-bored tumor cells during chemotherapy. The question iswhether melanoma cells can spontaneously spread into thethymus. A previous study has already demonstrated that B16melanoma growing under the skin of mice can metastasize tothe thymus in 6 of 20 mice (14).

Chemo/radiotherapy is a necessary adjunct treatement incancer therapy. However, this treatment not only kills cancercells, but also induces cancer stromal cell DDR to increaseinflammation. It was unclear which cell types are the mainsource of the thymic inflammation during chemotherapy. Basedon the B-lymphoma model in a previous report, it seems thatthe thymic inflammation arose from lymphoma cell deathinduced DDR (10). However, these lymphoma cells wereunable to circulate in great numbers into the thymus, and theydo not expand in the thymus. (We did not find thymoma

developed when we tested the lymphoma model; Supple-mentary Fig. S1 and data not shown.) Therefore, it is unlikelythat the majority of thymic inflammation comes from thesmall number of thymic-harbored tumor cell death. The inflam-mation was also proposed to come from thymic endothelialcells, associated with acute stress–associated phenotype (ASAP)-related secretion (11, 40). This is also unlikely because vascu-lature-related thymic endothelial cells represent very small por-tion, contained within 8% of the UEA-1�neg and Ly51�neg

subsets in �5% of the CD45�neg population, i.e., less than0.4% of total thymic cells based on a flow-cytometric assay(46). We find it hard to explain how so few cells could act as thepredominant source of thymic inflammation. The thymus is aunique organ and very senstive to any assault, especially tothe chemo/radiotherapy, which induces acute thymic atrophyasscociated with inflammation. Our data identified that underchemotherapy thymic inflammation arises from a synergisticeffect of cell death (mainly of thymocytes) and senescence(mainly of TECs), although we do not exclude cell death inTECs as well. In other words, the thymic inflammation arisesfrom chemotherapy-induced nonmaliganant thymic stromalcells, rather than the harbored tumor cells per se. The cell deathand senescence phenotype in thymic cells were mechanisticallydue to drug-associated DDR-triggered activation of the p53 gene,which induces both cell apoptosis (47) and cellular senescence(48, 49), which is illustrated in Supplementary Fig. S3.

Cellular senescence can be beneficial when it happens inmaliganat cells because it may arrest tumor cell growth, but it isdeleterious when it happens in nonmalignant stromal cells,because the cells then develop SASP-assocaited inflammation(39). TECs, mainly undergoing senescence during chemother-apy, are probably the cells involved in SASP-assocaited inflam-mation in the thymus. In addition, senescent stromal cellscould promote an epithelial-to-mesenchemal transition (38),which establishes a condition for cancer stem cell generationand cancer metastasis. As to inflammation, it is a double-edgedsword that can either to suppress tumor growth (such asimmune-mediated inflammation) or induce tumor cell pro-gression or chemoresistance (50–52).

The antiapoptotic feature with inactive state in thymic-har-bored tumor cells may reflect tumor cell dormancy at single-celllevels with intrinsic changes, such as increased activation of p38.These cells may display stem cell-like properties (termed "stem-ness"), which possess innate resistance mechanisms to chemo/radiotherapy (24). The reason may be due to epithelial-to-mesenchymal transition promoted by inflammation fromsenescent stromal cells (38). The risk is that these cancer cellseventually could develop into a tumor in distant organs (i.e.,metastatic relapse) once the conditons are suitable. In additon,we cannot exclude the possibility that some thymic-harboredcancer cells still retain sensitivity to chemotherapy, and theymay undergo a low balanced level of apoptosis and prolifera-tion to maintain nonreduced total tumor cell numbers in thethymus, which is termed "equal cell death." If this is the case, thethymic-harbored tumor cells exhibit a heterogeneous dormantstate, which makes these cancer cells neither likely to manifest asa tumor mass in the atrophied thymus, such as thymoma, nor tobe thoroughly eradicated in the atrophied thymus under thechemotherapy.

Taken together, our studies stand on the viewpoint that thebody's largest T lymphoid organ, the thymus, and especially the

Sizova et al.





chemotherapy-induced atrophied thymus, indeed provides achemoprotective microenvironment, playing the role of a cancerpremetastatic reservoir during chemotherapy. We brought a newtarget responsible for cancer relapse into focus. Considering thistarget during chemotherapy will potentially lead to efficienttherapeutic interventions to combat tumor recurrence.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: O. Sizova, D.-M. SuDevelopment of methodology: O. Sizova, D.-M. SuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): O. Sizova, D. Kuriatnikov, D.-M. SuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): O. Sizova, D. Kuriatnikov, D.-M. Su

Writing, review, and/or revision of the manuscript: O. Sizova, D.-M. SuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. LiuStudy supervision: D.-M. Su

AcknowledgmentsThe authors thank Dr. Alakananda Basu (Director of the Cancer Biology

Program at UNTHSC) and Rance Berg (Director and Graduate Advisor atUNTHSC) for critical reading of the manuscript and Dr. Xiangle Sun (CoreFacility at UNTHSC) for flow cytometer technical support. We also thank Dr.Michael T. Hemann (Department of Biology at MIT) for kindly providing theEm-Myc;p19Arf-/- B-cell lymphoma cell line.

This work was partially supported by The American Association of Immu-nologists (AAI) Careers in Immunology Fellowship Program, awarded toO. Sizova.

Received March 28, 2018; revised June 1, 2018; accepted June 22, 2018;published first July 13, 2018.

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2018;16:1652-1664. Published OnlineFirst July 13, 2018.Mol Cancer Res Olga Sizova, Denis Kuriatnikov, Ying Liu, et al. CellsAtrophied Thymus, a Tumor Reservoir for Harboring Melanoma

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Molecular Cancer Research - Atrophied Thymus, a Tumor … · The thymus was isolat-ed from cancer cell–inoculated mice and cut into tissue blocks. The tissue blocks were subcutaneously

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