SUMMARY Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from non-irradiated hosts. Molecular and genetic approaches show that TGFβ mediated tumor acceleration; molecular signatures implicated TGFβ and genetically reducing TGFβ abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGFβ independent and linked to mammary stem cell activity. Thus the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms.

Purpose: Several biomarkers of response to immune checkpoint inhibitors (ICI) show potential but are not yet scalable to the clinic. We developed a pipeline that integrates deep learning on histology specimens with clinical data to predict ICI response in advanced melanoma. Experimental Design: We used a training cohort from New York University (New York, NY) and a validation cohort from Vanderbilt University (Nashville, TN). We built a multivariable classifier that integrates neural network predictions with clinical data. A ROC curve was generated and the optimal threshold was used to stratify patients as high versus low risk for progression. Kaplan–Meier curves compared progression-free survival (PFS) between the groups. The classifier was validated on two slide scanners (Aperio AT2 and Leica SCN400). Results: The multivariable classifier predicted response with AUC 0.800 on images from the Aperio AT2 and AUC 0.805 on images from the Leica SCN400. The classifier accurately stratified patients into high versus low risk for disease progression. Vanderbilt patients classified as high risk for progression had significantly worse PFS than those classified as low risk (P = 0.02 for the Aperio AT2; P = 0.03 for the Leica SCN400). Conclusions: Histology slides and patients' clinicodemographic characteristics are readily available through standard of care and have the potential to predict ICI treatment outcomes. With prospective validation, we believe our approach has potential for integration into clinical practice.

Children exposed to ionizing radiation have a substantially greater breast cancer risk than adults; the mechanism for this strong age dependence is not known. Here we show that pubertal murine mammary glands exposed to sparsely or densely ionizing radiation exhibit enrichment of mammary stem cell and Notch pathways, increased mammary repopulating activity indicative of more stem cells, and propensity to develop estrogen receptor (ER) negative tumors thought to arise from stem cells. We developed a mammary lineage agent-based model (ABM) to evaluate cell inactivation, self-renewal, or dedifferentiation via epithelial-mesenchymal transition (EMT) as mechanisms by which radiation could increase stem cells. ABM rejected cell inactivation and predicted increased self-renewal would only affect juveniles while dedifferentiation could act in both juveniles and adults. To further test self-renewal versus dedifferentiation, we used the MCF10A human mammary epithelial cell line, which recapitulates ductal morphogenesis in humanized fat pads, undergoes EMT in response to radiation and transforming growth factor b (TGFb) and contains rare stem-like cells that are Let-7c negative or express both basal and luminal cytokeratins. ABM simulation of population dynamics of double cytokeratin cells supported increased self-renewal in irradiated MCF10A treated with TGFb. Radiation-induced Notch concomitant with TGFb was necessary for increased self-renewal of Let-7c negative MCF10A cells but not for EMT, indicating that these are independent processes. Consistent with these data, irradiating adult mice did not increase mammary repopulating activity or ERnegative tumors. These studies suggest that irradiation during puberty transiently increases stem cell self-renewal, which increases susceptibility to developing ER-negative breast cancer.

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.

SUMMARY A better knowledge of the regulatory mechanisms involved in stem cell proliferation and/or differentiation could reveal new methods for the treatment of some diseases. Most previous studies in the field of stem cell biology have been carried out on cultured isolated cells. In the case of adult tissue stem cells, mesenchymal bone marrow derived cells have been most widely studied, while the undifferentiated stem cells present in the epithelial tissues are less known. In order to advance further our understanding of epithelial tissue stem cells, new in vivo models are required. The present study focuses on the dynamics of a new and simple model of intestinal epithelial regeneration found in the midgut of the migratory locust, Locusta migratoria (Linnaeus 1758). The locust midgut consists of three cell types: columnar cells, endocrine cells and undifferentiated regenerative clustered cells. The undifferentiated epithelial midgut cells give rise to two other cell types and are located in a nest of regenerative cells known as regenerative niche. We have performed single and continuous bromodeoxyuridine (BrdU) administration experiments to study regeneration niches and their cellular dynamics. Immunocytochemistry and immunofluorescence techniques were used to detect the incorporation of BrdU into regenerative niches and the presence of FMRFamide-like immunoreactivity, as a marker for endocrine cell differentiation. Some isolated label retaining cells (LRC) were observed at the niche base 10-15 days after the final BrdU administration. We propose that these cells are the stem cells of this epithelial tissue. We also calculated the length of the cell cycle phases for a subpopulation of transit amplifying cells within the regenerative niche: G1, 2.5±0.5 h; S,5.5±0.5 h; G2, 0.75±0.25 h; M, 2.5±0.5 h. These amplifying cells will give rise to the columnar epithelial non-endocrine lineage. The differentiation of an endocrine cell from a niche stem cell occurs less frequently and thus leads to a lower proportion of endocrine cells as compared with epithelial columnar digestive cells (up to three endocrine cells per niche). Endocrine cell commitment seems to occur very early in the differentiation process within the niche, as double-labelled BrdU and FMRF endocrine cells have never been found. The only exception is the endocrine cells located in the ampullar region of the midgut, some of which show double immunostaining after long-term chronic BrdU injection. In summary, we have characterized a new and simple animal model of epithelial stem cell regeneration that may be useful for understanding the complex biological process that drives tissue renewal from undifferentiated and uncommitted progenitor cells.