Paraneoplastic renal dysfunction in fly cancer models driven by inflammatory activation of stem cells

Abstract

Tumors can induce systemic disturbances in distant organs, leading to physiological changes that enhance host morbidity. In Drosophila cancer models, tumors have been known for decades to cause hypervolemic 'bloating' of the abdominal cavity. Here we use allograft and transgenic tumors to show that hosts display fluid retention associated with autonomously defective secretory capacity of fly renal tubules, which function analogous to those of the human kidney. Excretion from these organs is blocked by abnormal cells that originate from inappropriate activation of normally quiescent renal stem cells (RSCs). Blockage is initiated by IL-6-like oncokines that perturb renal water-transporting cells, and trigger a damage response in RSCs that proceeds pathologically. Thus, a chronic inflammatory state produced by the tumor causes paraneoplastic fluid dysregulation by altering cellular homeostasis of host renal units.

Keywords: Biological Sciences; Drosophila; Physiology; Tumor-host; cancer model; kidney; paraneoplasia.

Figure 1:. Tumor-induced hypervolemia is associated with defective MT secretion

(A) Diagram of allografting into adult flies. (B-E) Flies grafted (g, dark spot shows injection site) with a WT disc appear normal (B), while flies grafted with a disc tumor show distended abdomens (C). In genetic OC cancer models, control adults raised at RT appear normal (D), while adults in which tumors are induced by a 29° shift develop abdominal swelling (E). Scale bar, 500 um. (F) The phenotype is due to fluid accumulation, as liquid weight is elevated in tumor-bearing flies. (G) Ex vivo assay demonstrates strongly compromised secretion of MTs isolated from tumor-bearing hosts. (H, I) The lower tubules (arrows) from control hosts are clear (H) while those from tumor-bearing hosts are opaque (I). Scale bar, 100 um.

Figure 2:. Tumor-bearing hosts accumulate excess cells in the renal tubule lumen

(A) Anatomy of MTs, the fly kidney analog (modified with permission from Ref. 21). Tubules absorb fluid from circulation and secrete it into a collecting duct; primary urine moves through the ureter for excretion. (B-F) In contrast to the patent lumen of control ureters shown by apical localization of VhaSFD (B, single confocal section), ureters from flies grafted with tumors are occluded by mispolarized cells (C). Control ureters (D, projected confocal stacks) show most nuclei of distinct small and larger sizes, reflecting RSCs and principal cells respectively. Imaging of tumor-bearing flies (E) shows an additional population of intermediate size (red arrows, quantitated in F). Scale bar in B, 100 um. Scale bar in B’ and D, 25 um. (G, H) The intermediate-size cells fill the ureter, as seen in cross-sections from control (G) versus tumor-bearing (H) flies. Scale bar, 25 um.

Figure 3:. Stem cell-derived progeny are required for fluid imbalance

(A-C) EdU incorporation demonstrates cell proliferation in tumor-bearing but not control MTs (A, B, quantitated in C). Scale bar, 100 um. (D-I) Ureter-blocking cells are derived from RSCs, as shown by lineage tracing driven by esg-GAL4 (D, E). However, these cells do not continue expression of the RSC marker esg-GAL4 (F, G); instead they express the PC marker Cut-GAL4 (H, I). Scale bar, 100 um. (J-P) Presence of RSCs in the MT is blocked by inhibiting Rac1 function during pupation (J, K). Scale bar, 100 um. When grafted with a tumor, these flies fail to develop hypervolemia (L, M, quantitated in N), contain MTs that show rescued fluid transport (O), and lack proliferating cells (P). Scale bar, 500 um.

Figure 4:. Ectopic STAT signaling in MTs of tumor-bearing hosts

(A-D) A reporter for STAT signaling, limited to RSCs (inset, with intensity elevated) in control MTs (A, B), is active in ureter-filling cells (C) as well as stellate cells in tumor-bearing hosts (D). Scale bar, 100 um. (E-J) Stellate cells of tumor-bearing flies lose their distinctive shape and reduce expression of markers such as Tsh>GFP (E, F, quantitated in G) and LKR-GFP (H, I, quantitated in J). Scale bar, 100 um.

Figure 5:. STAT activation in PCs drives renal obstruction

(A-F) STAT activation in stellate cells but not principal cells or RSCs is sufficient to cause fluid retention (A), as well as aberrant cells in the ureter (B, C) and defective stellate cells (D, E, quantitated in F). Scale bar, 100 um. (G) Hypervolemia is reduced when tumors are depleted of upd2 and upd3, with simultaneous expression of activated JAK to supply tumor-autonomous STAT activation. (H, I) Preventing STAT signaling in stellate cells through depletion of Dome reduces hypervolemia of tumor-bearing flies (H), quantitated in (I). Scale bar, 500 um. (J) The number of EdU-positive cells in the ureter of tumor-bearing hosts with/without Dome depletion in stellate cells. (K-M) Dome depletion also rescues stellate cell morphology and expression of LKR-GFP. Scale bar, 100 um.