Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly tumor support embryo life?

SUMMARY

The incidence of melanoma is increasing worldwide. It is one of the leading cancers in pregnancy and

the most common malignancy to metastasize to placenta and fetus. There are no publications about

experimental models of melanoma and pregnancy. We propose a new experimental murine model to

study the effects of melanoma on pregnancy and its metastatic process. We tested several doses of

melanoma cells until we arrived at the optimal dose, which produced tumor growth and allowed

animal survival to the end of pregnancy. Two control groups were used: control (C) and stress control

(SC). and three different routes of inoculation: intravenous(IV), intraperitoneal (IP) and subcutaneous

(SC). All the fetuses and placentas were examined macroscopically and microscopically. The results

suggest that melanoma is a risk factor for intrauterine growth restriction but does not affect placental

weight. When inoculated by the SC route, the tumor grew only in the site of implantation. The IP route

produced peritoneal tumoral growth and also ovarian and uterine metastases in 60% of the cases.

The IV route produced pulmonary tumors. No placental or fetal metastases were obtained, regardless

of the inoculation route. The injection of melanoma cells by any route did not increase the rate of fetal

resorptions. Surprisingly, animals in the IV groups had no resorptions and a significantly higher

number of fetuses. This finding may indicate that tumoral factors released in the host organism to

favor tumor survival may also have a pro-gestational action and consequently improve the

reproductive performance of these animals.

Title

Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly

tumor support embryo life?

Authors

Rubens H Bollos (Bollos, RH)a, Mary U Nakamura (Nakamura, MU)a, Valderez B V Lapchick

(Lapchick, VBL)b, Estela M A F Bevilacqua (Bevilacqua, E)c, Mariangela Correa (Correa, M)d, Silvia

Daher (Daher, S)a, Márcia M S Ishigai (Ishigai, MMS)e, Miriam G Jasiulionis (Jasiulionis, MG)d

a Department of Obstetrics, Sao Paulo Federal University Medical School, Sao Paulo, Brazil

b Animal Experimental Laboratory, National Institute of Pharmacology, São Paulo Federal University

Medical school, Sao Paulo, Brazil

c Department of Cellular Biology and Development, Institute of Biomedical Sciences, São Paulo

University, Brazil

d Department of Immunology, Sao Paulo Federal University Medical School , Sao Paulo, Brazil

e Department of Pathology, Sao Paulo Federal University Medical School , Sao Paulo, Brazil

Text

Cancer is the second cause of death during reproductive years and complicates 0.1% of all

pregnancies.[1-4] The most common malignancies associated with pregnancy include melanoma,

lymphomas, leukemias and cervical, thyroid and breast cancer. Melanoma is the most lethal skin

cancer and its incidence is increasing dramatically over the last decade worldwide.[5-7] Furthermore,

malignant melanoma is the 6th most frequently diagnosed cancer in women, affecting 35% of them in

reproductive phase (15-44 years).[8;9] Consequently, melanoma is one of the leading malignancies

associated with pregnancy, accounting for about 8% of all cancers diagnosed during gestation with

an incidence rate of 2.8 per 1000 deliveries.[3]

Cutaneous melanomas originate from individual melanocytes in about two-thirds of the cases and

from pre-existing cutaneous nevi in one-third. This tumor is aggressive with an unpredictable biologic

behavior. If detected early, surgical resection is often curative, and long-term survival is excellent.

Unfortunately, diagnosis is often delayed in pregnant women because changes in color or size of nevi

may occur during normal pregnancy. Therefore the disease is often diagnosed in more advanced

stages in these patients [10]. Hence, the recommendation that any patient presenting a suspicious

lesion or skin hyperpigmentation should always be submitted to a biopsy and histologically examined

as soon as possible. [11-13]

Melanoma accounts for more than 50% of all tumors in pregnancy that metastasize to placenta and

fetus.[14-18] Thus in these patients, both placenta and fetus should be examined thouroughly.[19;20]

Since it would be impossible to conduct prospective randomized clinical trials on the treatment of

melanoma in pregnancy, relevant data on therapy and management issues in pregnant women with

melanoma have been generated from case reports and matched historical cohort studies. Due to the

lack of sufficient data, it is difficult to draw firm conclusions about the effects of hormones and

pregnancy on melanoma, its prognosis and management in pregnant patients and also the safety of

prescribing oral contraceptives and hormone replacement therapy for patients with a history of

melanoma.[21;22] Most of the publications on the management and treatment of metastatic

melanoma in pregnancy have focused on the mother, with scarce analysis of placental and fetal

involvement.[23-25] Furthermore, the mechanism by which melanoma is able to overcome the

transplacental barrier is not well understood, and there are no well-established prognostic factors that

predict the increased probability of such fetal as placental metastasis.[26-28]

The objective of this study was to develop an experimental model for metastatic melanoma during

mice pregnancy. A second objective was to evaluate the effect of maternal melanoma on the fetus

and placenta, by morphological analysis.

METHODS

Cell line and culture

The murine melanocyte cell line melan-a[29] was cultured in RPMI pH 6.9 (Gibco, CA, USA),

supplemented with 5% fetal calf serum (Gibco) and garamicin (Invitrogen, CA, USA) at 37oC in a

humidified atmosphere of 5% CO2 and 95% air and 200 nM 12-o-tetradecanoyl PMA (Sigma, St.

Louis, MO). The murine melanomas cells lines Tm5[30;31] and B16F10 was cultured in the same

conditions, except for PMA. Cells were harvested after trypsin treatment of subconfluent monolayers

and counted, suspended in PBS in a concentration of 5.104cells in 100µl and injected as describe

below.

Animals

Sixty-eight female adult (8 weeks) virgin C57BL6 mice (Mus musculus domesticus), weighing

approximately 25 g, were included in the study. All animals were selected and maintained by the

Experimental Animal Laboratory of the Pharmacology Institute of São Paulo Federal University

(INFAR-UNIFESP). Animals were kept under 12h daylight cycles, with food and water ad libitum and

checked daily throughout the experiment. Females made estrous by induction, were caged overnight

with males (1:1) and successful mating was verified the following morning. The presence of a vaginal

plug indicated day “0” of pregnancy. All procedures were carried out in accordance with the

guidelines of the Brazilian College of Animal Experimentation and were authorized by the Committee

on Ethics in Animal Research - UNIFESP.

Groups and tumor cells inoculation

After mating, on day “0”, the 68 pregnant mice were randomly divided into 7 groups. Three groups

were inoculated with 5.104 Tm5 cells in 100µL PBS either subcutaneously (SC, n=10), intraperitonealy

(IP, n=10) and intravenously via tail vein (IVTm5, n=12). Two others groups were inoculated

intravenously with 5.104 B16F10(IVB16F10, n=8) and melan-a (IVmelan-a, n=8) cells. The control group

received no cells (C, n=10) and the stress control group received 100µL PBS intravenously (S, n=10).

Morphological assay

All animals were observed daily until the 19th day of gestation when they were euthanized by cervical

dislocation and submitted to a thoraco-abdominal laparotomy to expose internal organs. Various

maternal organs and tissues were carefully inspected to evaluate tumor dissemination. In each group,

fragments from different sites were collected for microscopic examination: the skin tumor from SC;

the peritoneum, uterus and ovaries from IP and the lungs from IV groups. The presence of tumor in

these organs meant successful cell implant. A uterine incision was then performed to extract the

fetuses and placentas. Resorptions were counted and weighed from each offspring. Maternal

organs and tissues as well as the fetal skin and placenta were examined under a stereoscopic

microscope in search of dark colored pigmented lesions. Placental tumoral involvement was defined

as gross or microscopic evidence of melanotic melanoma cells within any section of the placenta.

Similarly, fetal metastasis was defined as the presence of maternal neoplasic cells in the fetus, with

no evidence of a primary fetal tumor. Maternal organs, fetus, placenta and resorption tissues were

collected, analyzed, photographed and fixed n 10% para-formaldehyde in PBS 0.1 M, pH 7.2, for

morphological analysis.

Reproductive performance

The reproductive performance was assessed by fetal and placental weights and implantation and

resorption rates. Implantation rate was defined as the number of fetus/pregnant animal and resorption

rate as the number of resorption/pregnant animal.

Morphological analysis and histochemistry assay for identification of metastasis

All placentas, fetuses and resorbed embryos were sliced sagitally . Representative fragments from

maternal organ were embedded in paraffin and processed. The blocks were sliced using a microtome

adjusted at 3 µm. The slices were placed on glass slides previously lubricated with Mayer albumin.

The slides were left in an oven at 37ºC for 24 hours to dry and fix the material. All the slides

(maternal and fetal organs, placenta and resorptions) were then stained with hematoxylin-eosin (HE)

and Fontana-Masson[32] (which provides differential staining of melanotic cells). Two slides from

each blocks were examined by two observers using a conventional light photomicroscope (Olympus,

Optical Co. Ltd., Tokyo, Japan). All fields in each section were evaluated (x40, x100 and x400) for the

presence of pigmented areas and anaplasic cells. Representative microphotographs of the maternal,

fetal, placental and resorption tissues were obtained.

Statistical analysis

Numerical data, number of fetuses, resorbed embryos and the weights of placentas and fetuses were

expressed as means ± standard deviation, medians, minimum, maximum values. Differences among

the groups were analyzed and compared using the Kruskal–Wallis test (H). The Dunn’s test for

multiple comparisons was used when significant differences were observed. A 3.03 version of the

GraphPad TM software was used for this purpose. p < 0.05 was taken as statistically significant..

RESULTS

Maternal survival after tumor inoculation

Female mice were inoculated with different doses of melanoma cells via the subcutaneous route to

ascertain time to tumor progression and death (Figure 1). For gestational studies, 5.104 Tm5 cells

was chosen as the best inoculation dose for all inoculation routes, since mice injected with

subcutaneous tumors could carry a pregnancy to term (around 20 days). All inoculated pregnant

mice (100%) survived until the 19th gestational day, with no apparent fetal preterm death.

Tumor and metastasis presence versus routes of cell inoculation

To analyze the impact of different forms of tumor presentation on the pregnancy outcome, we chose

3 routes for tumor inoculation. The SC route, which most closely resembles the natural history of

melanoma, the IV route to simulate metastatic disease and the IP route to simulate pelvic organ

invasion. Microscopic analyses revealed viable melanoma cells in all pregnant mice (n=40),

regardless of the inoculation route. As shown in Figure 2A, 2B and 2C, all animals presented

subcutaneous tumors when inoculated via this route (n=10). Similarly, the injection of two tumor cells

intravenously (n=20) resulted in metastatic disease in all lungs study animals (Figure 2J,2K,2L,2M).

The injection of melan-a non-tumorigenic cell line intravenously (n=8) resulted in no tumor growth, as

expected (Figure 2N,2O). Locally invasive disease was found in the peritoneum surface (Figure

2D,2G,2H) in all mice injected via the intraperitoneal route (n=10). In this last group, ovarian and

uterine metastasis were identified in 80% and 20% of the animals, respectively (Figure 2E,2F, 2I).

Placental and fetal metastasis detection

A total of 800 slides from placentas and fetuses (C=78, IVTm5=170, IVB16F10=138, IVmelan-a=114, IP=82,

SC=114, S=104) were stained by hematoxylin-eosin (HE) and analyzed by conventional light

microscopy. Because throphoblast and melanoma cells share the expression of several antigens

used for melanoma immunological diagnosis (S100, Melan-A and HMB-45), the histochemical

method using the Fontana-Mason staining solution was chosen for melanoma and melanin pigment

detection to confirm HE findings. Although an extensive analysis was performed, no evidence of

metastatic disease to either the fetuses or placental tissues was observed (data not shown).

Implantation and resorption rates analysis

The number of implantations and resorptions was determined by direct visual examination of the

uterus. The implantation rate was significantly higher (p<0.001) in the IVTm5, IVB16F10 and IVmelan-a

groups and the resorption rate was significantly lower (p<0.005) just in the IV melanomas groups,

compared to all other groups, include IVmelan-a. In the IP and SC groups, 20% of pregnant mice had

only resorptions and no live fetuses (Table1, Figure 3).

Fetal and placental weight measurements

Fetal weight was negatively influenced by tumor cell injection by any route, and also by sham venous

puncture, suggesting that the stressful conditions of the experiment were responsible for this findings.

Unexpectedly, placental weight was not altered by any experimental conditions (Table 2, Figure 3).

DISCUSSION

Malignant melanoma accounts for more than 50% of all the tumors that metastasize to the fetus or

placenta. This tendency to metastasize to these structures is intriguing and poorly understood[33-35]

In combination with the fetal immune system, the placental barrier is remarkably efficient at

preventing the spread and allografting of the maternal tumor into the fetus and the transmission of

neoplastic and nonneoplastic maternal cells to the fetus that occurs during pregnancy involves only

aggressive and rapidly growing tumors such as melanomas and hematological malignancies.[36]

Alternatively the point in placental/fetal development at which they are exposed to maternal tumor

cells may be critical in terms of tumor tolerance or rejection.[37]Thus, once separation of the fetal and

maternal blood systems is breached and maternal intravascular tumor cells can pass through the

placenta, they commonly manifest initially as skin lesions or abdominal swelling and end up in the

fetal liver via the umbilical vein or in the fetal lungs via the ductus venosus or placental

implants.[38;39] In this experimental model we searched for metastatic cells in these fetal organs,

both macroscopically and microscopically. Due to its enormous blood supply, it can be difficult to

identify metastatic cells within the placenta, even using microscopy.[40;41] Because of this, in this

experiments we decided to examine not only tissue fragments but the entire placenta and fetus and

use both HE and a specific melanotic stain (Fontana-Masson). Despite all this, no melanoma cells

were identified in any of the fetuses or placentas in all groups.

Fetal metastases require maternal tumor invasion and fetal inability to eliminate metastatic cells.

According to the literature, fetuses who develop melanoma metastases may have developed

immunologic tolerance because of exposure to tumor antigens at a time when the developing

immune system is not yet capable of responding.[42;43] One hypothesis is that the fetus rejects

maternal malignant cells as a homograft. Fetal T- and B-cell responses are thought to develop

around weeks 7 -10 and 14 -20, respectively, and most of the reported cases of fetal metastases

occurred in women who had metastatic disease before that period. Thus early fetal metastases,

before a critical phase of immunologic maturation of the fetus, might be the result of an inability to

recognize the tumor cells as foreign. Moreover the timing of maternal metastasis of melanoma does

not seem to differ in placental versus fetal metastasis.[44;45] In reviews, male infants seem to be at

higher risk than females for developing metastases of any maternal cancer. Males comprise 80% of

all infants with metastasis of melanoma and 75% with metastasis of all cancers indicating that gender

may influence survival.[46-50] A possible explanation is that female fetuses are more capable of

eliminating maternally derived melanoma or that male fetuses are more immunotolerant.[51;52] This

fact is very interesting since embryogenesis and consequently the genetic sex, is controlled by both

genetic and epigenetic mechanisms.[53-55]. Epigenetics and DNA methylation are being held

responsible for the appearance of cancer and cause strong and heritable transcriptional silencing of

genes subject to genomic imprinting or to X chromosome inactivation. Inactivation provides a unique

model for studying the establishment and maintenance of chromosome-wide gene silencing and

alterations in that mechanism could explain the high frequency of melanoma cell in male fetus.[56]

Since in our experiment there were no fetal metastasis we did not evaluate the gender of the fetuses.

The most likely way for dissemination of cancer by vertical transmission is through the hematogenous

route[57-59]. Because of this, we inoculated melanoma cell during mice pregnancy by three different

routes (IP, SC and IV) in order to produce vertical transmission or transplacental metastases. For

metastatic assay (IV) we inoculated two tumorigenic lineages (Tm5 and B16F10) and non-

tumorigenic cell line (melan-a). Despite the fact that all mothers developed melanoma and none of

them died, no tumoral cells implants were found in the fetuses or placentas. Ovarian (Figure 2E,2F)

and uterine (Figure 2I) metastases were identified in the IP group, demonstrating that this was an

adequate model for research in female intrapelvic organ metastases. Despite the lack of fetal or

placental metastasis, melanoma cells injected subcutaneously, intraperitoneally or intravenously

reduced fetal weight (Table 2, Figure 3). This suggests that metastatic maternal melanoma may have

a deleterious effect on embryo implantation and fetal development. The SC group mimics the natural

history of melanoma and in this group we found the highest resorption rates (Table 2, Figure 3). This

could perhaps indicate the presence of factors that inhibit trophoblast invasion and embryo

implantantion. Alternatively the poor reproductive performance could be the result of caquexy induced

by rapid tumoral growth, which eventually will lead to maternal death. It is possible that cytokines

involved in the process of caquexy (tumor necrosis factor alpha, interleukins 1, 2 and 6 and gama

interferon) could be implicated[60;61]. Melanomas release these cytokines recognizably involved in

the TH1 immunological response, which antagonizes trophoblast invasion and embryo

implantation.[62;63] These cytokines are also involved in missed abortions and placental diseases

such as gestational trophoblastic tumors, pre-eclampsia and intra-uterine growth retardation[64-66].

The high resorption rate could indicate the decline in maternal health due to disease spread with

consequent embryo death. In the IVTm5 and IVB16F10 groups lung metastases (Figure 2J,2K,2L,2M)

developed as expected in all mothers from the both melanomas groups. No metastasis or cell

developted in the lung of IVmelan-a group (Figure 2N,2O). As previously discussed, the complete

absence of resorptions and the elevated number of live fetuses were very significant (Table 1, Figure

3). These findings suggest that, contrary to the other groups, the intravenous inoculation of tumor

cells may actually favor trophoblast implantation and embryo development. Hence we hypothesize

that once melanoma metastatic cells reach the maternal blood stream produce a release of various

factors, as cytokine and growth factors and their receptors wich by autocrine and paracrine effect

enable them to grow autonomously and confer competence to metastasis. Autocrine growth factors

as basic fibroblast growth factor (bFGF), melanoma growth stimulatory activity (MGSA), platelet-

derived growth factor (PDGF)-A , stem cell growth factor, IL-8 and IL-10 are produced by melanocyte

and melanoma cells wich act on cell proliferation.[67;68] The growth factors produced by melanoma

cells as epidermal growth factor(EGF), transforming growth factor (TGF-β), (GM-CSF), insulin-like

growth factor-1 (IGF-1), nerve growth factor (NGF), hepatocyte growth (HGF), vascular endothelial

growth factor factor (VEGF) can have paracrine effects on other cells in the surrounding stroma while

the endothelial cell, fibroblasts, monocytes, lymphocytes wich are involved in tumor growth and

invasion.[69] Autocrine and paracrine effects could explain the beneficial effects on embryo and

placental development by melanoma cells observed in IVTm5 and IVB16F10 groups (Table 1). Also in

IVmelan-a group we observed this benefit effect by elevated number of fetuses once melanocytes cell

can express many of these factors during their development by autocrine regulation. Oppositely, the

number of resorptions was elevated in this group (Table 1), revealing that just malignant cells

produce excessive levels of autocrine and paracrine grow factors and proliferate autonomoysly and

infinitevely[70].

As part of the metastatic model recently proposed[71] metastatic cells must alter the expression of

numerous genes, including expression or repression of specific cell adhesion molecules, elaboration

of matrix-digesting enzymes, expression of proto-oncogene products, activation of telomerase and

the acquisition of a rich blood supply before they are able to invade and establish themselves in an

alternative tissue environment. Under this model many similarities exist between metastatic cancer

cells implantation and embryo implantation. The trophoblast cells of the placenta proliferate, migrate

and invade the pregnant uterus and its vasculature in order to nourish the developing fetus in a way

that is imitated by malignant tumors.[72;73] The placenta is an exceptionally vascular organ and

produces several growth factors (placental growth factor, HGF, VEGF and tissue factor). These

placental angiogenic factors also have been reported to be released by and influence the growth of

melanoma cells in culture.[74;75] Also expression of endothelial intercellular adhesion molecules

(ICAM), in particular ICAM-1 and the upperegulation of N-cadherin, α5β3 integrin and

downregulation of E-cadherin and α6β1 integrin, encourage adhesion, survival and invasion of

melanoma cells and metastatic spreading to distant sites. However, the precise mechanism by which

these growth factors contribute to these processes has yet to be defined and more research is

needed. Furthermore, some studies have shown that the thickness of melanomas diagnosed during

pregnancy is significantly greater than that of site matched melanomas in non-pregnant women in the

same age range.[76-79] This may be due to growth factors released during normal pregnancy which

may offer a selective growth advantage to the developing malignant melanocytes. This could also

justify the relative affinity of melanomas for placental and fetal metastasis once the risk of vertical

transmission in mothers with placental metastasis involvement is 25%.[80;81]

The similarities between trophoblast invasion and metastatic cells bring to mind the phenomenon of

multiple or simultaneous cancers. In humans and a few animals, a second tumor may sometimes

arise at another location, within a short time after the appearance of the first malignancy. In such

cases, studies have reported hat the first cancer immunologically enhances the growth of other

cancers from premalignant cells.[82-84] This led us to wonder if the development of one tumor

(melanoma) could facilitate the outgrowth of another “tumor” (trophoblasts). The higher rate of

implantation and absence of resorptions in the IV groups seem to indicate this could have occurred

in those animals. However, the other groups, which received the same melanoma cells by other

routes had a poor reproductive performance (Table 1). This may suggest that perhaps pregnancy

outcome may be influenced not only by the presence of tumor cells but also by the route of

inoculation. These findings are very intriguing and further questions have been formulated.

In summary, an experimental model for the study of metastatic melanoma during mice pregnancy

was developed. Surprisingly, the intravascular inoculation of melanoma cells in pregnancy seems to

favor trophoblast and embryo development. These findings may contribute to our understanding of

the mechanisms involved in trophoblast invasion and embryo implantation. Furthermore, these

findings may also be potentially useful in the development of new therapies for immunological

miscarriages, placental diseases and assisted reproduction.

Acknowledgements

This work was supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de

São Paulo) and CAPES. We thank Drs. Gilberto Costa Freitas and Maria Regina Torloni for reviewing

the manuscript and offering helpful comments.

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Competing interests statement: The authors declare no competing financial interests.

Table 1.

Table 1 - Implantation and resorption rates analysis

group implantation

rate resorption

rate

control (C)

n = 10

3,9 ± 1,37 3,5

2 / 6

1,5 ± 0,71 1,5

1 / 2

stress (S)

n = 10

5,2 ± 1,48 5,5

3 / 7

2 ± 1 2

1 / 3

subcutaneous (SC)

n = 10

5,7 ± 2,91 6

0 / 10

3,5 ± 1,87 3,5

1 / 6

intraperitoneum (IP)

n = 10

4,1 ± 2,89 5

0 / 8

2,5 ± 1,29 2,5

1 / 4

intravenous (IVTm5) n = 12

7,08 ± 1,38

7*

5 / 10

0 ± 0

0*

0 / 0

intravenous (IVB16F10)

n = 8

8,6 ± 0,91

9*

7 / 10

0 ± 0

0*

0 / 0

intravenous (IVmelan-a)

n = 8

7,1 ± 1,12

7* 6 / 9

2,5 ± 1 2

1 / 4

datas are :

mean ± s.d. median

min./ max

* Kruskal–Wallis test showed significant differences among the groups p<0,001

Table 2.

Table 2 - Fetal and placental weight measurements

group placenta weight

fetus weight

control (C)

n = 10

0,12 ± 0,02 0,13

0,08 / 0,15

1,06 ± 0,04

1,08* 0,99 / 1,11

stress (S)

n = 10

0,13 ± 0,007 0,13

0,12 / 0,14

0,99 ± 0,03 0,99

0,95 / 1,03

subcutaneous (SC)

n = 10

0,13 ± 0,005 0,13

0,12 / 0,14

0,99 ± 0,02 0,99

0,96 / 1,03

intraperitoneum (IP)

n = 10

0,13 ± 0,01 0,12

0,11 / 0,16

0,99 ± 0,02 0,99

0,94 / 1,03

intravenous (IVTm5) n = 12

0,12 ± 0,01 0,12

0,11 / 0,14

0,99 ± 0,01 0,99

0,97 / 1,01

intravenous (IVB16F10)

n = 8

0,13 ± 0,01 0,13

0,12 / 0,14

0,99 ± 0,02 0,98

0,97 / 1,02

intravenous (IVmelan-a)

n = 8

0,12 ± 0,01 0,13

0,11 / 0,14

0,95 ± 0,04 0,98

0,94 / 1,01

datas are :

mean ± s.d. median

min./ max

* Kruskal–Wallis test showed significant differences among the groups, p<0,005

0 2 4 6 8 10 12 14 16 18 200

20

40

60

80

1002.105 NG

2.105 G

105 G

5.104 G

p<0,05

days after inoculation

% t

um

or-

free a

nim

als

A

0 2 4 6 8 10 12 14 16 18 20 22 240

20

40

60

80

100

2.105 NG

2.105 G

105 G

5.104 G

p<0,05

days of inoculation

% s

urv

ival

B

Figure 1 legend. Tumoral doses versus tumoral growth and maternal survival after tumor

implant: the figure (A) displays the time needed for tumoral growth after the inoculation of melanoma

cells. The subcutaneous route was used for inoculation in the first studies for this model. Females

were inoculated on day “0” of pregnancy (vaginal plug). The 5.104 dose produced palpable tumoral

growth (>20mm3) in all mice within 16 days and allowed maternal survival in good conditions beyond

the end of pregnancy (20th day), showing be the best dose as figure (B) depicts.

Figure 2 legend. Morphology assay: (A) skin tumor (arrow) in pregnant mouse of SC group, (D)

melanoma implants in epiploon (arrow) after IP inoculation of melanoma. (G) Resorption (black

arrow) in IP group and melanoma metastasis on the epiploon (white arrows). (J) Black dots (arrow)

represent lung metastases after IV inoculation of melanoma cells. Uterus and fetuses exposed.

Photomicrograph of histological analysis of tissue sections from the skin (B,C) in SC group; ovaries

(E,F), epiploon (H) and uterus (I) metastasis of mouse from IP group and lung metastasis (K,L,M)

and lung after IV inoculation of melanocyte cell stained by Fontana-Mason histochemistry

(E,H,I=x40);(C,K,O=x100);(F,M=x400) and Hematoxylin-Eosin (B,L,N=x100).

D A G J

K E B

H

L C F I

M

N

O

C S SC IP IVTm5 IVB16F10 IVmelan-a0

2

4

6

8

10

12increase of implantation rate in IV group

* **

*p<0,0001

nu

mb

er

of

fetu

s

A

____C S SC IP IV

Tm5IV

B16F10IV

melan-a0

1

2

3

4

5

6

7

*p<0,005

absence of reabsortion in IV melanoma groups

**

nu

mb

er

of

rea

bs

ort

ion

B

C S SC IP IVTm5 IVB16F10 IVmelan-a

0.95

0.97

1.00

1.02

1.05

1.07

1.10

1.12

*fetal under weight by melanoma and stress

*p<0,005C

we

igh

/g

C S SC IP IVTm5

IVB16F10

IVmelan-a

0.07

0.09

0.11

0.13

0.15

0.17placental weight was not change by tumor

p=ns

we

igh

t/g

D

Figure 3 legend. Boxplots of reproductive maternal performance in experimental model of

metastasis in mice pregnancy by inoculation of Tm5 melanoma cells via intraperitoneum (IP),

subcutaneous (SC) and inoculation of Tm5, B16F10 and non-tumorigenic melan-a cell intravenous

(IV) routes. Two control groups were used: stress group for puncture (S) and pregnant control (C). In

the IV groups there was an increased number of fetuses (A) and no resorptions (B) just in

melanomas groups, suggesting that intravenous injection of tumoral cells could support trophoblast

implantation and fetal development. Tumor and stress produced reduction of fetal weight (C) but did

not affect placental weight (D). * P-values were computed using the Kruskal-Wallis test.

Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly tumor support embryo life?

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