In vivo and in vitro evolution of small cell lung cancer molecular subtypes under cytotoxic treatment

Abstract

Introduction

Small cell lung cancer (SCLC) is an aggressive form of lung cancer with a high mortality rate. There is currently a “one-size fit all” strategy relying on chemotherapy plus immunotherapy. The emergence of a classification of SCLC into 4 subtypes, based on the expression of transcription factors (ASCL1, NEUROD₁and POU2F3 or triple negative) [1], opens the way to personalised therapeutic strategies according to subtype-specific vulnerabilities [2]. Consequently, we sought to evaluate in vitro and in vivo the evolution of subtypes under chemotherapy.

Methods

Two complementary models have been developed to test our hypothesis:

1) in vitro: exposing SCLC lines (H₄₄₆ NEUROD₁ +  H₆₉ ASCL1+ and H₅₂₆ POU2F3 + ) to carboplatin. The evolution of subtypes has been evaluated both at protein level using western blots and RNA level using qPCR.

2) in vivo: analysing CDXs (CTC-Derived Xenografts) tumours before and after exposure to chemotherapy. Cytotoxic drugs tested were carboplatin alone (n = 4), combination of carboplatin and etoposide (n = 4) and lurbinectedin (n = 6). The expression of the different subtypes was characterised on tumours by immunohistochemical (IHC) staining using H-score.

Results

We observe a decrease of NEUROD₁ protein expression after exposure of H₄₄₆ cells to carboplatin. This phenotype was detectable only after 3 days of chemotherapy exposure and was independent of NEUROD₁ mRNA level. Similarly, we observe a decrease of ASCL1 and POU2F3 protein expression after carboplatin treatment in H₆₉ and H₅₂₆ cells, respectively. In CDXs models, we observed a global decrease of ASCL1 expression both after lurbinectedin and carboplatin/etoposide combination in the 54.01 CDX tumors, and a strong decrease of NEUROD₁expression after carboplatin in the 51.01 CDX tumors. Noticeably, POU2F3 positive cells emerge in hostpots within tumour samples after lurbinectedin treatment.

Conclusion

We demonstrate using both in vitro and in vivo models a decrease of neuro-endocrine transcription factors ASCL1 and NEUROD₁ under cytotoxic-pressure. The mechanisms responsible for those changes are still to be explored. This concept of cellular plasticity modulated by chemotherapy paves the way for therapeutic sequence guided by tumour's phenotype.