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Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a high mortality rate. The presence of a dense desmoplastic stroma rich in fibroblasts, extracellular matrix, and immune cells plays a critical role in disease progression, therapy response and is a distinguishing feature of PDAC. PDAC is currently treated with a combination of surgery, chemotherapy and radiation therapy in selected cases which results in long-term survival only in a small percentage of patients. Cancer therapies that incorporate immunotherapy-based techniques have become increasingly common in recent years. While such a strategy has been shown to be effective for immunogenic, “hot” tumors like melanoma and lung cancer, thus far PDAC patients display poor responses to this therapeutic approach. Various factors, such as low tumor mutational burden, increased infiltration of immunosuppressive cells, like MDSCs and Treg cells promote tolerance and immune deviation, further aggravating adaptive immunity in PDAC. In this review we will elaborate on the ability of PDAC tumors to evade immune detection. We will also discuss various 3D model system that can be used as a platform in preclinical research to investigate rational combinations of immunotherapy with chemotherapy or targeted therapy, to prime the immune microenvironment to enhance antitumor activity.

SummaryOvarian cancer metastasizes into the peritoneum through dissemination of transformed epithelia as multicellular spheroids1, 2. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues3: lumen surrounded by smoothly contoured, adhered, and immotile epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid ‘moruloid’ clusters that permit intercellular movement and penetration by new cells. Moruloid clusters can coalesce to form bigger clusters. Upon further culture, moruloid clusters mature into ‘blastuloid’ spheroids with smooth contours, lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow penetration by new cells. Ultrastructural examination reveals a basement membrane-like matrix coat on the surface of blastuloid, but not moruloid, spheroids: immunocytochemistry confirms the presence of extracellular matrix proteins: Collagen IV and Laminin-322. Enzymatic debridement of the coat results in a reversible loss of lumen and contour. Debridement also allows spheroidal coalescence and cell intrusion in blastuloid spheroids and enhances adhesion to peritoneal substrata. Therefore, the dynamical matrix coat regulates both the morphogenesis of cancer spheroids and their adhesive interaction with their substrata, affecting ultimately the progression of the disease.ResultsSurvival of women afflicted with epithelial ovarian cancer (EOC) trails behind other gynecological malignancies, despite improvements in surgical-pharmacological approaches4,5. The morbidity associated with the disease is a consequence of its transcoelomic route of metastasis: transformed epithelia of the fallopian tubes and ovaries in the form of spheroids, eventually home and adhere to the mesothelial lining of the peritoneum, occasionally invade through the underlying collagenous extracellular matrix and form secondary metastatic foci around abdominal organs1,6, 7. EOC spheroids impede the drainage of the fluid from the peritoneal cavity and alter its composition; in turn the fluid, now known as malignant ascites serves as a pro-tumorigenic milieu for the spheroids8, 9The formation and presence of spheroids within ascites of an ovarian cancer patient is strongly associated with recurrence of cancer and greater resistance to chemotherapy10. Therefore, in order to develop novel strategies to target spheroidal metastatic niche, it is essential to investigate mechanisms that underlie their morphogenesis. Several proteins have been proposed to mediate the adhesion between ovarian cancer epithelia that give rise to spheroids. These include transmembrane receptors such as CD4411, cell adhesion molecules, such as E-cadherin and N-cadherin12, matrix adhesion-inducing proteins such as integrins13, 14. Remarkably, a phase-contrast microscopic examination of spheroids from patients, or from aggregated epithelia of immortalized cancer lines cultured on low attachment substrata, shows features of morphogenetic organization: presence of a central lumen, radially arranged apposed epithelia and compacted surfaces. Such traits are cognate to organized morphogenesis within the glandular epithelial organs,15which are built through principles that include, but are not limited to, cell-cell adhesion16, 17. In fact, loss of tissue architecture seen in tumorigenesis involves the disappearance of such morphogenetic traits (such as matrix adhesion and polarity)18, 19.In this manuscript, we investigate how these traits are recapitulated in a fluid metastatic context. Using spheroids from patients with high grade serous adenocarcinoma and ovarian cancer cell lines, we show that the development of a basement membrane (BM)-like coat of extracellular matrix is responsible for the compaction and stability of cancer spheroids, for decreasing the motility of cells within it and for generation of lumen. The coat, which is rapidly replenished by cells upon enzymatic debridement, also prevents the attachment of spheroids to matrix substrata. This may have significant implications for the build-up of the massive cellular fraction within the malignant ascites of patients afflicted with ovarian cancer.

Expression dynamics of basement membrane-like extracellular matrix and fibronectin regulate transitions between solid (moruloid) and cavitational (blastuloid) phenotypes of ovarian cancer spheroids., Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid “moruloid” clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into “blastuloid” spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.