BackgroundThe lifesaving chemotherapy and radiation treatments that allow patients to survive cancer can also result in a lifetime of side-effects, including male infertility. Infertility in male cancer survivors is thought to primarily result from killing of the spermatogonial stem cells (SSCs) responsible for producing spermatozoa since SSCs turn over slowly and are thereby sensitive to antineoplastic therapies. We previously demonstrated that the cytokine granulocyte colony-stimulating factor (G-CSF) can preserve spermatogenesis after alkylating chemotherapy (busulfan).MethodsMale mice were treated with G-CSF or controls before and/or after sterilizing busulfan treatment and evaluated immediately or 10–19 weeks later for effects on spermatogenesis.ResultsWe demonstrated that the protective effect of G-CSF on spermatogenesis was stable for at least 19 weeks after chemotherapy, nearly twice as long as previously shown. Further, G-CSF treatment enhanced spermatogenic measures 10 weeks after treatment in the absence of a cytotoxic insult, suggesting G-CSF acts as a mitogen in steady-state spermatogenesis. In agreement with this conclusion, G-CSF treatment for 3 days before busulfan treatment exacerbated the loss of spermatogenesis observed with G-CSF alone. Reciprocally, spermatogenic recovery was modestly enhanced in mice treated with G-CSF for 4 days after busulfan. These results suggested that G-CSF promoted spermatogonial proliferation, leading to enhanced spermatogenic regeneration from surviving SSCs. Similarly, there was a significant increase in proportion of PLZF+ undifferentiated spermatogonia that were Ki67+ (proliferating) 1 day after G-CSF treatment.ConclusionsTogether, these results clarify that G-CSF protects spermatogenesis after alkylating chemotherapy by stimulating proliferation of surviving spermatogonia, and indicate it may be useful as a retrospective fertility-restoring treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0226-1) contains supplementary material, which is available to authorized users.

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Low dose computed tomography (LDCT) is the standard of care for lung cancer screening in the United States (US). LDCT has a sensitivity of 93.8% but its specificity of 73.4% leads to potentially harmful follow-up procedures in patients without lung cancer. Thus, there is a need for additional assays with high accuracy that can be used as an adjunct to LDCT to diagnose lung cancer. Sputum is a biological fluid that can be obtained non-invasively and can be dissociated to release its cellular contents, providing a snapshot of the lung environment. We obtained sputum from current and former smokers with a 30+ pack-year smoking history and who were either confirmed to have lung cancer or at high risk of developing the disease. Dissociated sputum cells were counted, viability determined, and labeled with a panel of markers to separate leukocytes from non-leukocytes. After excluding debris and dead cells, including squamous epithelial cells, we identified reproducible population signatures and confirmed the samples’ lung origin. In addition to leukocyte and epithelial-specific fluorescent antibodies, we used the highly fluorescent meso-tetra(4-carboxyphenyl) porphyrin (TCPP), known to preferentially stain cancer (associated) cells. We looked for differences in cell characteristics, population size and fluorescence intensity that could be useful in distinguishing cancer samples from high-risk samples. We present our data demonstrating the feasibility of a flow cytometry platform to analyze sputum in a high-throughput and standardized matter for the diagnosis of lung cancer.

Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso‐tetra (4‐carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin‐mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly (KD=42nM) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.