Epithelial Regeneration After Doxorubicin Arises Primarily From Early Progeny of Active Intestinal Stem Cells

Abstract

Background & Aims aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood. Methods We monitored DXR’s effect on aISCs by enumerating Lgr5-eGFP + and Olfm4 + crypts, cleaved caspase-3 (CASP3 + ) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations ( Bmi1 + , Hopx + , Dll1 + , and Defa6 + ) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR. Results Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3 + cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3 + cells). There was minimal regenerative contribution from Bmi1 + , Hopx + , Dll1 + , and Defa6 + -expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU + , and expressed low levels of DDR transcripts. Conclusions We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs’ robust DDR, which facilitates expeditious apoptosis.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury

“…Accordingly, recent studies support a novel paradigm in which the progeny of Lgr5+ CBCs after chemical and radiation injury undergo de-differentiation to reconstitute fresh Lgr5+ CBCs and regenerate the epithelium [5][6][7] .…”

Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells

“…We collected all tissues at 3 days (72 hours) post DXR as the maximal depletion of aISCs is observed by this time point post DXR in GF ( Figure 1b ) and CONV mice ( Figure 2a ). 6

Figure 2. Antimicrobial-treated mice retain aISCs after DXR.

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“…We hypothesized that the microbiota present at the time of injection would be the most influential on the aISCs, as these cells are expulsed from the crypt base as early as 24 hours after DXR injection. 6 Therefore, we focused on the influence of AMBx treatment in the jejunum. We compared the differential expression of OTUs between non-AMBx and AMBx mice, without the confounding influence of DXR ( Figure 6d ).…”

“… 4 , 5 Recent work from our lab demonstrates acute loss of aISCs in response to doxorubicin in conventionally raised (CONV) mice with wild-type microbiota. 6 …”

Lgr5+ intestinal stem cells are required for organoid survival after genotoxic injury

“…DXR targets proliferating cells by inhibiting topoisomerase II and evicting histones (Pang et al, 2013). In the intestine, this primarily affects aISCs and results in fISC-driven regeneration (Jones et al, 2019; Sheahan et al, 2021). We reasoned that an optimized DXR injury assay in organoids could model injury-induced cycles of aISC/fISC fate transitions in an epithelial autonomous manner.…”