Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. PDAC is characterized by a complex tumor microenvironment (TME), that plays a pivotal role in disease progression and resistance to therapy. Investigating the spatial distribution and interaction of TME cells with the tumor is the basis for understanding the mechanisms underlying disease progression and represents a current challenge in PDAC research. Imaging mass cytometry (IMC) is the major multiplex imaging technology for the spatial analysis of tumor heterogeneity. However, there is a dearth of reports of multiplexed IMC panels for different preclinical mouse models, including pancreatic cancer. We addressed this gap by utilizing two preclinical models of PDAC: the genetically engineered, bearing KRAS–TP53 mutations in pancreatic cells, and the orthotopic, and developed a 28–marker panel for single–cell IMC analysis to assess the abundance, distribution and phenotypes of cells involved in PDAC progression and their reciprocal functional interactions. Herein, we provide an unprecedented definition of the distribution of TME cells in PDAC and compare the diversity between transplanted and genetic disease models. The results obtained represent an important and customizable tool for unraveling the complexities of PDAC and deciphering the mechanisms behind therapy resistance.

BRCA1 deficiency is observed in approximately 25% of triple-negative breast cancer (TNBC). BRCA1, a key player of homologous recombination repair, is also involved in stalled DNA replication fork protection and repair. Here, we investigated the sensitivity of BRCA1-deficient TNBC models to the frequently used replication chain terminator gemcitabine, which does not directly induce DNA breaks. A large fraction of BRCA1-deficient cells was sensitive to gemcitabine, in contrast to their isogenic BRCA1-proficient counterparts. Gemcitabine-treated BRCA1-deficient cells accumulated massive levels of ssDNA and presented no RPA or RAD51 nuclear foci. The gemcitabine-induced accumulation of ssDNA in BRCA1-deficient cells was strongly diminished by targeting MRE11 with inhibitors and by siRNA attenuation. In contrast, treatment with the PARP1/2 inhibitor olaparib did not result in MRE11-dependent over-resection. Furthermore, a fraction of gemcitabine-treated BRCA1-deficient cells that showed massive ssDNA accumulation slipped into mitosis, producing mitotic bridges and strongly stained bromodeoxyuridine (BrdU) and γH2AX micronuclei. The BrdU-positive micronuclei and DNA bridges also stained positively for cyclic GMP–AMP synthase. In conclusion, these data suggest that gemcitabine treatment in BRCA1-deficient TNBC exposes unprotected nascent DNA linked to replication fork reversal, which leads to MRE11 over-resection and ssDNA accumulation. Therefore, the observed hypersensitivity to gemcitabine indicates that it could be a beneficial addition to BRCA1-deficient TNBC treatment. Significance: Treating BRCA1-deficient triple-negative breast cancer with gemcitabine induces massive ssDNA production due to MRE11 over-resection and causes cells to slip into mitosis, produce micronuclei, accumulate DNA breaks, and ultimately die.

BRCA1 deficiency is observed in approximately 25% of triple-negative breast cancer (TNBC). BRCA1, a key player of homologous recombination repair, is also involved in stalled DNA replication fork protection and repair. Here, we investigated the sensitivity of BRCA1-deficient TNBC models to the frequently used replication chain terminator gemcitabine, which does not directly induce DNA breaks. A large fraction of BRCA1-deficient cells was sensitive to gemcitabine, in contrast to their isogenic BRCA1-proficient counterparts. Gemcitabine-treated BRCA1-deficient cells accumulated massive levels of ssDNA and presented no RPA or RAD51 nuclear foci. The gemcitabine-induced accumulation of ssDNA in BRCA1-deficient cells was strongly diminished by targeting MRE11 with inhibitors and by siRNA attenuation. In contrast, treatment with the PARP1/2 inhibitor olaparib did not result in MRE11-dependent over-resection. Furthermore, a fraction of gemcitabine-treated BRCA1-deficient cells that showed massive ssDNA accumulation slipped into mitosis, producing mitotic bridges and strongly stained bromodeoxyuridine (BrdU) and γH2AX micronuclei. The BrdU-positive micronuclei and DNA bridges also stained positively for cyclic GMP–AMP synthase. In conclusion, these data suggest that gemcitabine treatment in BRCA1-deficient TNBC exposes unprotected nascent DNA linked to replication fork reversal, which leads to MRE11 over-resection and ssDNA accumulation. Therefore, the observed hypersensitivity to gemcitabine indicates that it could be a beneficial addition to BRCA1-deficient TNBC treatment.Significance:Treating BRCA1-deficient triple-negative breast cancer with gemcitabine induces massive ssDNA production due to MRE11 over-resection and causes cells to slip into mitosis, produce micronuclei, accumulate DNA breaks, and ultimately die.