To assess the involvement of ERRa in bone metastasisformation, CT (pool of CT-1 and -2 clones), WT-1, and AF2 (pool of AF2-1, -2 and -3 clones) cells were inoculated intra- venously into female BALB/c nude mice. Thirty-five days after tumor cell injection, radiographic analysis revealed that ani- mals bearing WT-1 tumors had osteolytic lesions that were 40% smaller than those of mice bearing CT tumors (Fig. 3A,B, and J). By contrast, there was a 3-fold increase in the extent of osteolytic lesions in animals bearing AF2 tumors, when com- pared with control (Fig. 3A, C, and J). The inhibitory effect of ERRa on cancer-induced bone destruction was confirmed using 3D micro-CT reconstruction (Fig. 3D–F), histology (Fig. 3G–I), and histomorphometric analyses of tibiae (BV/ TV; skeletal tumor burden, TB/STV; Fig. 3J). Taken together, our results indicated that overexpression of ERRa in breast cancer cells reduced the formation of osteolytic lesions. Regulation of OC formation by ERRa#expressing BO2 cells
Impact of vascular PPAR beta/delta expression on tumor progression and metastasisformation
Peroxisome proliferator- activated receptors (PPARs) are nuclear receptors and function as transcriptional factors involved in some chronic diseases such as lipid metabolism disorder and cancer, which include PPAR alpha, PPAR beta/delta, PPAR gamma. Among them, PPAR beta/delta has exhibited explicitly proangiogenic effects on physiological and pathological angiogenesis. Angiogenesis has been known as a hallmark of cancer. PPAR beta/delta has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is still unclear to what extent the expression of PPAR beta/delta in tumor endothelium influences tumor progression and metastasisformation. Thus, we addressed this question using transgenic mice with conditional inducible vascular-endothelium specific PPAR beta/delta overexpression. We also induced syngenic tumors following specific PPAR beta/delta overexpression in endothelial cells. We observed higher tumor neovascularization and enhanced tumor growth and metastasisformation in tumors of animals with vascular-specific PPAR beta/delta overexpression. In order to identify potential downstream molecular targets of PPAR beta/delta in tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet- derived growth factor B (PDGFB), platelet- derived growth factor receptor beta (PDGFR beta) and the receptor tyrosine kinase KIT (c-KIT) as new PPAR beta/delta dependent molecules by ChIP assay. Thus, we demonstrated that PPAR beta/delta activation, regardless of its action on different cancer cell types, promotes tumor progression and metastasisformation through enhancement of tumor angiogenesis.
The inability of the merging theory to explain all of the observed volumes may indicate that besides merging by passive motion due to proliferation, other mechanisms such as chemokine- mediated cells attraction occur [ 6 , 50 ]. Circulating tumour cells may be attracted by some estab- lished niches and explain the abnormally fast volume expansions that we observed. Indeed, such chemokine-mediated attractions are presumed to play an important role for the pre-met- astatic and metastatic niches establishment, in mediating myeloid and tumour cells attraction [ 6 , 50 , 51 ]. Moreover, chemo-attractants may play a role in tissue tropism of metastatic cells [ 52 ]. Chemotactic gradients can attract metastatic cells that express the chemokine receptor to specific locations. In the future, additional phenomena such as aggregation and recruitment of cells during the metastatic process from the circulation should be integrated in the standard mathematical model. Another phenomenon that could possibly explain the observed volumes would be the presence of circulating tumour cell clusters that would give rise to metastases [ 32 ]. Indeed, Aceto et al. recently showed in a breast cancer animal model that metastases do not originate from single cells only but also from tumour cells clusters that have a higher meta- static potential than single cells. However, they did not show evidence of this phenomenon for kidney cancer and in their experiments, clusters were formed by at most 50 cells. As indicated above, this order of magnitude of the initial cell numbers that colonizes the lung is not able to describe the dynamics of metastasisformation in our model and experimental data.
initiating ALDH high cells and contain a mixture of cell types  . Indeed, FACS analyses revealed that MMTV-PyMT;Tgfbi D/D tumours have reduced num- bers of ALDH high cells and express lower levels of Ald- h1a3 (Fig. 1D and Fig. S1 f,g). Accordingly, tumour cells derived from TGFBI-deficient mice have 37 times less tumour-initiating capacity in limiting dilution assays (Fig. 1E ). In addition, MMTV-PyMT;Tgfbi D/D tumours also showed a significant reduction in meta- static Lin CD24 + CD90 + cells (Fig. 1F ) and conse- quently seeded less lung metastases (Fig. 1G ). These results were confirmed by lung metastasis assays, which demonstrated that the metastatic capacity of the tumour cells is determined by their origin, regardless of the genotype of the host in which they were injected (Fig. 1H ). However, sphere, tumour and metastasisformation were not significantly decreased or increased by either knocking out or overexpressing Tgfbi in breast cancer cells (Fig. S2 ). These results indicate that TGFBI-driven microenvironmental changes in the pri- mary tumour influence the CSC phenotype. Taken together, these data suggest that TGFBI in the ECM is an important driver of tumour- and metastasis-initi- ating capacity in breast cancer.
In the present work, we demonstrate that LLC-lung metastasisformation is enhanced in DUSP3 -/- mice compared to DUSP3 +/+ littermates. This phenomenon was however specific to LLC and E0771 cells since no difference of B16 metastatic dissemination was observed between DUSP3 +/+ and DUSP3 -/- mice. Studies have reported that reduced vascular permeability led to a decrease in metastasis after LLC and B16 i.v. injection [ 30 , 31 ]. Although vascular permeabil- ity is significantly enhanced in DUSP3 -/- mice when compared with DUSP3 +/+ mice (unpub- lished observations), the fact that B16-induced metastasis was not influenced by DUSP3 deletion rules out the possible involvement of vascular permeability in the observed pheno- type. The second plausible premise could be that LLC and E0771-metastatic cells respond dif- ferently to the DUSP3 -/- tumour microenvironment. A differential regulation of experimental LLC and B16 metastasisformation has been previously shown in Nrf2-deficient mice. Indeed these mice developed lung metastasis faster compared to control mice upon LLC but not B16 cells i.v. injection [ 32 ]. In the case of LLC metastasis, tumour formation was associated with a higher recruitment of immune cells (MDSC). The authors of the study concluded that Nrf2 facilitates appropriate immune responses against LLC cells and therefore plays an anti-meta- static role and that the phenotype was restricted to the lung microenvironment [ 32 ]. The dif- ferential involvement and regulation of immune cells was further confirmed by the fact that the recruitment of myeloid-derived suppressor cells and dendritic cells are different in LLC and B16 experimental metastasis [ 33 ]. In our model, the role of immune cells was clearly dem- onstrated by the use of bone marrow chimeric mice. The transplantation of DUSP3 -/- bone marrow cells into irradiated DUSP3 +/+ mice accelerated the development of metastatic in recipient mice since DUSP3 -/- -> DUSP3 +/+ mice developed significantly larger lung metasta- sis compared to control mice, demonstrating that the hematopoietic compartment is responsi- ble for the increased LLC metastasis in DUSP3 -/- mice.
activity (Fig. 2A, right). Metastatic cancer cells were injected intracardiacally into the bloodstream of nude mice to reca- pitulate the late, rate-limiting, steps of the metastatic process, and examine metastatic dissemination to various organs while avoiding any effect of ATIP3 on primary tumor growth. Four groups of 18 mice were analyzed in two independent experi- ments. For each animal, the total number of metastatic foci and the number of photons/s were quantiﬁed every 2 days for 24 days (Supplementary Table S3). As shown in Fig. 2B, the time-course of metastasisformation was markedly delayed in mice injected with ATIP3-positive as compared with ATIP3- negative cell clones. The number of cancer cells growing at secondary sites increased exponentially from day 17 after injection of ATIP3-positive clones, as compared with day 10 for mice injected with control cells (Fig. 2B). As shown in Fig. 2C, the number of mice developing metastasis was strongly diminished upon ATIP3 expression. Importantly, the number of detectable metastases per mouse was also signiﬁcantly reduced at all times in the presence of ATIP3 (Fig. 2D). At day 24, the number of mice invaded with large metastases reached 13 of 18 (72.2%) in the control group as compared with 2 of 18 (11.1%) following injection of ATIP3-positive cells (Fig. 2E and F), indicating a prominent effect of ATIP3 on cancer cell growth and colonization at secondary sites. Accordingly, on day 24, the total number of photons/s per mouse was 50- and 25-fold lower following injection with Cl3 and Cl6 clones, respectively, compared with WT (Supplementary Fig. S2A). For ethical reasons, mice had to be sacriﬁced at day 24, therefore OS of the two groups of mice could not be quantiﬁed. Furthermore, ex-vivo and histologic analysis of metastatic nodules (Supplementary Fig. S2B) conﬁrmed that biolumines- cent signals indeed correspond to metastases of human tumor cells having inﬁltrated mouse tissues. Metastases were mainly detected in the bones, the lungs, and the brain, which are the most frequent sites of metastatic dissemination of human breast tumors. No preferential location of metastatic nodules in ATIP3-positive versus ATIP3-negative cell types could be observed. Altogether, these results identify ATIP3 as a potent antimetastatic molecule, and support a role for ATIP3 in metastatic growth and colonization in vivo.
general increases in sialylation on metastatic cell lines(51), our study suggests a conserved mechanism by which the T-Antigen is presented on metastasizing cells that appears to potentiate colonization through interactions with galectins in the metastatic niche. It is worth noting that a recent study found that another sialyltransferase, ST6GalNAcII, may influence metastatic potential (52). In this case, however, knockdown of ST6GalNAcII in a breast cancer cell line led to an increase in metastasis. While, on the surface, this finding appears to contradict those presented within this manuscript, the data are consistent in that in both cases, glycosylation-dependent galectin-3 binding promotes metastasisformation. One possibility is that the inverse regulation of the two transferases may result in the same functional phenotype, with ST6GalNAcII preventing presentation of the galectin-3 ligand N-Acetyllactosamine or similar ligands, and ST6GalNAcIV promoting expression of the T-Antigen. Previously, we found that the integrin α3β1 promotes metastasis through binding to combinations of
If the prevalence of EMT phenotypes in clusters is so far largely uncovered, it is nevertheless recognized that CTC clusters contain hybrid E/M phenotypes. Thus, single-cell RNA sequencing of clustered CTCs versus isolated CTCs from breast cancer patients has revealed that several cell–cell contact molecules are overexpressed/maintained in clusters, such as plakoglobin, which has been further shown to be functionally involved in cluster metastatic potential [ 151 ]. Complicating the theory and blurring the border between E and M states even further, EMT/CSC markers may also be implicated in cell–cell interactions in clusters. Accordingly, CD44, which has been consistently reported on CTCs in most types of cancer [ 50 , 100 , 187 , 188 ], was recently shown to contribute to intravascular aggregation of tumor cells through the formation of homophilic intercellular interactions [ 153 ]. CD44 + cell aggregates were also found to be more resistant to apoptosis than single cells. Other recognized markers of mesenchymal or CSC phenotypes have also been detected in CTC clusters in vitro or in mouse models such as Tenascin C or Jagged1 [ 152 , 189 ]. A recent study of CTCs isolated from breast cancer patients and mouse models, reported that the binding sites for CSC transcription factors such as OCT4, SOX2, or NANOG, are hypomethylated in CTC clusters compared with isolated CTCs [ 49 ]. Other data collected from human samples also emphasize the presence of hybrid E/M phenotypes in heterogeneous CTC clusters, particularly in lung cancers [ 51 , 52 , 109 , 190 ]. Very elegantly, a study by Yu and coworkers identified cells expressing both epithelial (such as EpCAM or cytokeratins) and mesenchymal markers (including fibronectin, N-cadherin or PAI-1) in isolated CTCs but also in CTC clusters from breast cancer patients [ 179 ].
Qiu et al. identiﬁed a physical interaction between PD-L1 and H- Ras, which led to Ras/Erk activation. In glioma cells, this activation promoted EMT, migration, and invasion [ 96 ]. Xue et al. studied glioma specimens and found a correlation between PD-L1 expression and ma- lignancy features, like the expression of proangiogenic VEGF and the proliferative marker Ki-67 [ 97 ]. When PD-L1 was silenced in melanoma cells, Clark et al. showed reduced metastasis in an in vivo mouse model [ 98 ]. In DM, a rare subtype of melanoma with frequent local re- currences, PD-L1 played a role in promoting cell proliferation [ 99 ]. DM is a unique form of melanoma, characterized by a prominent host re- sponse and high PD-L1 expression. Those authors found a positive correlation between PD-L1 and p53 expression, the Ki-67 proliferation index, tumoral and stromal CD8 T cell in ﬁltration, and stromal Treg inﬁltration. They also found that PD-L1 expression in cancer was cor- related with tumor thickness, mitosis, recurrence, and metastasis [ 99 ]. In CRPC, ATM is an apical activator of the DNA damage response [ 100 ]. This kinase showed signiﬁcantly enhanced expression in prostate cancer tissues from patients with CRPC, and high ATM levels were as- sociated with EMT features and high PD-L1 levels [ 100 ]. An ATM knockout in CRPC cells resulted in reduced PD-L1 levels. The same eﬀect on PD-L1 was obtained when cells were treated with a JAK in- hibitor; this treatment resulted in a signi ﬁcant reduction in the ex- pression of EMT-associated genes [ 100 ]. These ﬁndings suggested that the ATM/JAK/PD-L1 axis played a role in the transition from hormone dependence to hormone independence in prostate cancer, and also in the progression of CRPC cells to metastasis [ 100 ]. The relevance of the ATM/JAK/PD-L1 axis in the induction of EMT was further assessed in
46. Hiratsuka S, Watanabe A, Aburatani H, Maru Y: Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol 8: 1369-1375, 2006
47. Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C, MacDonald DD, Jin DK, Shido K, Kerns SA, Zhu Z, Hicklin D, Wu Y, Port JL, Altorki N, Port ER, Ruggero D, Shmelkov SV, Jensen KK, Rafii S, Lyden D. VEGFR1- positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438: 820-827, 2005
A significant number of patients in the U.S with metastatic brain tumors face a dismal prognosis and high mortality. Increasing numbers of breast cancer patients are being diagnosed with brain metastases, possibly as a result of the emergence of targeted and aggressive systemic cancer therapy. In overall frequency, breast cancers and lung can- cers are by far the most common cancers that metastasize to brain [1,2]. Brain metastasis generally arises in women diagnosed with aggressive breast cancer or in men with advanced lung cancer. The actual incidence of brain metastases is not precisely known, however studies sug- gest that 6–16% of patients with metastatic breast cancer develop brain metastases during their lifetime. Further- more, autopsy studies have reported brain metastases in 18–30% of patients dying ofbreast cancer . The major- ity of women who develop brain metastases have pre- sented with debilitating neurological symptoms, and have undergone aggressive treatment for stage IV disease [4-6]. Although brain metastasis is the leading cause of breast cancer death, its pathogenesis is poorly understood and the predictors of breast metastasis to brain are yet to be characterized.
BMI1 overexpression promotes melanoma cell invasion and distant site colonization
Having shown that BMI1 promotes metastases formation, we assessed BMI1 ’s ability to modulate specific stages of the metastatic cascade. First, we examined migration us- ing wound healing assays (for A375, B16F0, B16F1, and B16F10 BMI1 vs. CTL cells, and MA2 and B16F10 sh-Ctl vs. sh-BMI1 lines) (Fig. 2A; Supplemental Fig. S2A) and transwell migration assays (for A375, S91, and B16F10 BMI1 vs. CTL cells) (Supplemental Fig. S2B). Migration was significantly increased by BMI1 in all cases and signif- icantly decreased with the highest degree of BMI1 knock- down (sh#2 for MA2 and sh#1 for B16F10). We then used an in vivo extravasation assay to assess BMI1 ’s contribu- tion to metastasis seeding. BMI1 or parental B16F10 cells were labeled with a red fluorescent dye (CMRA) and in- jected into the tail veins of nude mice (n = 3 for each vari- ant and time point). Two hours later, equivalent numbers of BMI1 and parental cells were found in the lungs, indi- cating comparable capillary entrapment (data not shown). In contrast, 48 h after injection showed a significant in- crease (1.6-fold) in lung occupancy of BMI1 versus paren- tal cells (Fig. 2B). Importantly, we confirmed that the CRMA-labeled tumor cells had exited the blood vessels (detected by CD31 immunostaining) and colonized the lung parenchyma (Supplemental Fig. S2C). We then used in vitro extravasation assays to compare the ability of BMI1 and CTL A375 cells to invade through an endothe- lial cell monolayer (Fig. 2C). Again, the BMI1 cells migrat- ed significantly better than CTL cells (+35%; P = 0.001), showing that BMI1 enhances extravasation in a cell- autonomous manner.
intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells’ elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis.
. Nevertheless, the molecular intermediates regulat- ing the protumoral effects of AhR deficiency could not be determined.
In this study, we have found that Aldh1a1 upregula- tion is likely an intermediate factor promoting melan- oma growth and metastasis in AhR depleted cells. Consistent with that hypothesis, AhR knockdown failed to exert a pro-tumoral effect when Aldh1a1 was simul- taneously inactivated. Interestingly, depletion of basal Aldh1a1 levels in AhR-expressing melanoma cells did not significantly affect tumor growth, suggesting that the overactivation of Aldh1a1 is likely a causal factor increasing the tumorigenicity of AhR deficient melan- oma cells. Therefore, the tumor suppresor role of AhR in melanoma  could take place by antagonizing the Aldh1a1 activity. We suggest that the coordinated expression of AhR and Aldh1a1 could be a useful molecular marker in melanoma.
We also demonstrate in vivo that stimulation of some ORs expressed in tumor cells could facilitate cells dissemination and metastasis generation. Indeed, we stimulated xenografted LNCaP cells in NSG mice with a PSGR ligand. Without stimulation, mice developed tumors at 85% of the inoculation sites, but also some metastases in inguinal nodes, spine and liver, which is seldom reported in the literature concerning prostate cancer models using immunodeficient mice and LNCaP cells [37–39]. When treated with mineral oil alone or containing b-ionone, mice showed a significantly increased number of metastases, despite the small number of animals in the experiment. Noteworthy, only the b- ionone treated mice developed metastases in other tissues, namely in lungs and Tyson glands, and those in Tyson glands were particularly well developed. In addition, we showed that the mineral oil used as an excipient in our experiments induces LNCaP cell invasiveness by activating the PSGR and involving a PI3Kc inhibitor pathway, like b-ionone. These results corroborate that metastases emergence enhancement observed in mineral oil treated mice can be due to PSGR activation, at least partly, since we cannot exclude the presence of other ORs in LNCaP cells and of their specific ligands in the mineral oil. It would be interesting to identify the mineral oil component activating the PSGR, but this appears difficult due to the complex and unavailable detailed composition of mineral oil. Moreover, while in vitro there was no additive effect of mineral oil and b-ionone in promoting cell invasion, in vivo, addition of b-ionone to mineral oil not only slightly boosted metastasis emergence in the same tissues but moreover induced metastasis spreading to additional tissues. Also, mice treated with b-ionone had to be sacrificed earlier due to faster tumor growth, and the number of metastases detected in
3. Epithelial–Mesenchymal Transitions: Impact on CTC Phenotypic Heterogeneity
EMTs have long been known as crucial actors in metastasis. The examination of EMT actors in CTCs has thus logically gained rapidly growing interest in the past decade [ 68 – 74 ].
The generally accepted view [ 75 – 81 ] is that EMTs generate various hybrid phenotypes along the epithelial (E) to mesenchymal (M) differentiation axis, thereby contributing importantly to tumor heterogeneity. If most epithelial and mesenchymal states are believed to harbor limited metastatic potential, certain E/M hybrid phenotypes are considered to harbor high degree of epithelial–mesenchymal plasticity (EMP), enabling them to undergo timely and spatially regulated dynamic and reversible interconversions within a “plasticity window”. These phenotypical adaptations are crucial for tumor cells to survive/develop in the different microenvironments encountered during the metastatic spread. After an eventual period of dormancy, a switch towards more epithelial proliferative states (mesenchymal–epithelial transitions, METs) is further suspected to occur during metastatic outgrowth. EMTs would therefore rather be involved in the initial steps of the metastatic spread: entry in the circulation, survival in the bloodstream, arrest on the vasculature, and early phases of metastatic niching [ 68 , 75 – 83 ]. Whether the same hybrid tumor cell is able to overcome all obstacles of the metastatic cascade through phenotypic adaptations or whether further genetic alterations occur during the metastatic cascade that empower some tumor cells to form metastases is still a subject of debate. Cooperative processes between different phenotypes may also occur, by which EMT-shifted cells would help more epithelial phenotypes (with higher competence for metastatic outgrowth) to gain and survive in the circulation, and niche in secondary organs [ 84 , 85 ].
RT-PCR analysis of cancer-cell-specific markers (Pymt; cyto- keratin 8 [Krt8]) in the blood revealed that fewer cancer cells circulated in PyMT +/ mice ( Figures 1 G and S1 G). This was not
due to a difference in survival of cancer cells in the blood, as veri- fied by an in vitro clonogenic assay (mimicking non-adherent conditions in the blood) ( Figure 1 H), suggesting reduced cancer cell intravasation. PHD2 haplodeficiency did not reduce metas- tasis by affecting cancer cell extravasation and colonization, as analyzed respectively by RT-PCR of genes involved in extrava- sation ( Figure S1 H) or Pymt in the lungs after intravenous injec- tion of PyMT +/ or PyMT +/+ cancer cells in wild-type recipient mice ( Figure S1 I). Thus, global PHD2 haplodeficiency impairs metastasis without affecting primary tumor growth in a sponta- neous tumor model.
Fig. 2 Intratracheal injection of CCL11 recombinant (recCCL11) induces 4T1 tumor cell migration to lung tissue (n = 6/group). a Timeline of
experiment. Mice were challenged with i.t of recCCL11 and i.v injected with luciferase-stably transfected 4T1 cells. b, c On day 15, biophotonic monitoring of lung metastasis in animals and quantification of bioluminescence in regions of interest (ROI) determined around lungs. d, e Representative hematoxylin–eosin–stained sections of lung tissues and tumor size quantification assessed by measuring the ratio between the area of tumor foci in lungs and total lung tissue area on 8 sections per mouse in each group. Scale bar: 2.5 mm, 100 µm. Results are expressed as mean tumor area/lung area ± SEM. **p < 0.01
Umbilical skin metastases (or Sister Mary Joseph nodules) are rare. Their presence typically indicates the late manifestation of deep-seated abdominopelvic malignancy. They occur mainly in gynecological cancers, and gastrointestinal cancers in men. The most common his- tology is adenocarcinoma (∼75% of cases), but it can also rarely be squamous cell or undif- ferentiated carcinoma. These metastases can be present at diagnosis or appear at disease recurrence, and are associated with a very poor prognosis with an average survival of 11 months. We report the clinical case of a 58-year-old man with metastatic pancreatic adeno- carcinoma and umbilical cutaneous metastasis after receiving first-line chemotherapy. The diagnosis was established upon liver biopsy in July 2019, after the patient presented with a complaint of transfixing abdominal pain. The first-line treatment consisted of six cycles of modified FOLFIRINOX chemotherapy. However, in November 2019, computed tomography (CT) scan showed disease progression. Second-line treatment with gemcitabine (Gemzar®) led to a 16% decrease in target lesions. During the fourth cycle, three periumbilical indurated