Circulating Tumor Cells (CTCs) in blood encompass DNA, RNA, and protein biomarkers, but clinical utility is limited by their rarity. To enable tumor epitope-agnostic interrogation of large blood volumes, we developed a high-throughput microfluidic device, depleting hematopoietic cells through high-flow channels and force-amplifying magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from seven patients with metastatic cancer. High CTC yields (mean 10,057 CTCs per patient; range 100 to 58,125) reveal considerable intra-patient heterogeneity. CTC size varies within patients, with 67% overlapping in diameter with WBCs. Paired single-cell DNA and RNA sequencing identifies subclonal patterns of aneuploidy and distinct signaling pathways within CTCs. In prostate cancers, a subpopulation of small aneuploid cells lacking epithelial markers is enriched for neuroendocrine signatures. Pooling of CNV-confirmed CTCs enables whole exome sequencing with high mutant allele fractions. High-throughput CTC enrichment thus enables cell-based liquid biopsy for comprehensive monitoring of cancer.

We previously demonstrated that Bim is the main BH3-only protein replacing Bak/Bax from Bcl-xl to activate apoptosis in a p53-independent manner in response to doxorubicin in prostate cancer. However, the comparison of doxorubicin treatment between LNCaP cells carrying p53-wild type and PC3 cells carrying p53-null suggested that p53 might be essential for maximizing apoptosis. Inhibition of ATM did not affect doxorubicin-induced apoptosis. Overexpression of p53 did not affect ABT-263-induced apoptosis and nevertheless, the combination of doxorubicin with ABT-263 induced higher apoptotic responses than did doxorubicin or ABT-263 alone. These results advocated that doxorubicin-induced DNA damage controls p53 function for intensifying apoptosis. Indeed, overexpression of p53 only enhanced apoptosis under conditions of severe DNA damage induced by high concentrations of doxorubicin in LNCaP cells. Immunofluorescence staining showed vague γH2AX foci and enlarged nuclei in LNCaP cells in response to high concentrations of doxorubicin, en route to apoptosis. In addition, our results revealed that the apoptosis in response to DNA replication stress induced by CFS-1686, a catalytic inhibitor of topoisomerase, is p53-independent. Interestingly, the combination of doxorubicin with CFS-1686 generated DNA damage and replication stress simultaneously, resulting in a synergistic apoptotic effect in prostate cancer cells. Thus, we concluded that p53 is a sensor for enhanced apoptosis in response to DNA damage stress, not DNA replication stress, at least in prostate cancer.

We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.

Doxorubicin and other anthracycline compounds exert their anti-cancer effects by causing DNA damage and initiating cell cycle arrest in cancer cells, followed by apoptosis. DNA damage generally activates a p53-mediated pathway to initiate apoptosis by increasing the level of the BH3-only protein, Puma. However, p53-mediated apoptosis in response to DNA damage has not yet been validated in prostate cancers. In the current study, we used LNCaP and PC3 prostate cancer cells, representing wild type p53 and a p53-null model, to determine if DNA damage activates p53-mediated apoptosis in prostate cancers. Our results revealed that PC3 cells were 4 to 8-fold less sensitive than LNCaP cells to doxorubicin-inuced apoptosis. We proved that the differential response of LNCaP and PC3 to doxorubicin was p53-independent by introducing wild-type or dominant negative p53 into PC3 or LNCaP cells, respectively. By comparing several apoptosis-related proteins in both cell lines, we found that Bcl-xl proteins were much more abundant in PC3 cells than in LNCaP cells. We further demonstrated that Bcl-xl protects LNCaP and PC3 cells from doxorubicin-induced apoptosis by using ABT-263, an inhibitor of Bcl-xl, as a single agent or in combination with doxorubicin to treat LNCaP or PC3 cells. Bcl-xl rather than p53, likely contributes to the differential response of LNCaP and PC3 to doxorubicin in apoptosis. Finally, co-immunoprecipitation and siRNA analysis revealed that a BH3-only protein, Bim, is involved in doxorubicin-induced apoptosis by directly counteracting Bcl-xl.

The bispecific antibody tarlatamab recruits T cells to cancers expressing the neuroendocrine epitope delta-like ligand 3 (DLL3). Tarlatamab is effective in small cell lung cancer (SCLC), but clinical outcomes vary, and no biomarkers enable patient selection. Single-cell RNA sequencing of SCLC biopsies identifies heterogeneity in DLL3 expression, and analysis of circulating tumor cells (CTC) distinguishes individual patients as predominantly DLL3Pos or DLL3Low. In a prospective cohort of 20 patients, pretreatment DLL3 expression on CTCs predicts tarlatamab clinical benefit (85% sensitivity and 100% specificity). Necrotic CTC clusters in blood accompany treatment-induced tumor lysis. Acquired resistance to tarlatamab is associated in some cases with loss of DLL3 expression but persistence of other targetable neuroendocrine epitopes; in other patients, DLL3 is retained on CTCs but accompanied by systemic markers of T-cell dysfunction. Quantitation of DLL3-positive CTCs identifies patients likely to benefit from tarlatamab, and longitudinal monitoring may guide therapeutic decision-making at the time of acquired resistance. Significance: CTCs are abundant in SCLC, allowing noninvasive quantitation of epitopes targeted by immune therapies. Patients with at least 25% DLL3-positive CTCs derive clinical benefit from a bispecific antibody targeting this epitope; those with a lower fraction rarely respond. CTC scoring may thus enable stratification of patients for antibody-mediated therapeutics.