Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis

Abstract

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

Keywords: Prussian Blue; clear cell; ferroptosis; iron; metastasis; renal cell carcinoma.

Figure 1

Kidney cancer has high levels of iron compared with other common cancers. The incidence and mean tissue level (H-score) of non-heme iron staining was compared across a variety of cancer types using two different TMA sources: (A, C) the RPCCC multi-cancer TMA that included primary tumor tissue from 14 different body sites and (B, D) the Biomax multi-cancer TMA that included primary tumor tissue from 10 different body sites. The number of patients evaluated for each tumor type in the RPCCC TMA is indicated above the error bar, and eight patients were evaluated for each tumor type in the Biomax TMA. (E) Representative staining images are shown at low power magnification for (left to right) kidney cancer (RCC), lung cancer, liver cancer, and hemochromatosis liver as a positive staining control for iron overload. *p < 0.01 vs. all other cancers; **p < 0.001 vs. all other cancers.

Figure 2

Increased iron levels in primary renal tumors and metastases. Prussian Blue staining for total iron was performed using the RPCCC RCC patient TMA set. (A) Iron staining incidence was compared among normal (non-neoplastic) kidney, renal primary tumors, and RCC metastases. (B) Mean iron levels (H-score) were compared among normal (non-neoplastic) kidney, primary tumors, and RCC metastases. (C) Iron staining incidence was compared among renal cell primary tumor histologic subtypes. (D) Mean iron levels (H-score) were compared among renal cell primary tumor histologic subtypes. (E) Representative images of iron staining from different kidney tissue types from left to right; top row: normal (non-neoplastic) kidney, renal oncocytoma, ccRCC, and hemochromotosis liver (positive control stain for iron overload); bottom row: papillary RCC, chromophobe RCC, and ccRCC metastasis. *p < 0.05; ***p < 0.001.

Figure 3

Iron levels in primary tumors decrease with tumor progression. Primary tumor iron levels (H-score) were evaluated using Prussian Blue stain of the RPCCC RCC patient TMA set and tested for association with pathologic features of primary tumors including (A) tumor size (largest diameter), (B) tumor stage, (C) tumor grade, and (D) presence of sarcomatoid dedifferentiation. Representative tissue core images are shown for low stage/grade (left) and high grade/stage (right) primary tumors of patients with (E) ccRCC and (F) pRCC. (G) Iron staining level (H-score) for the ccRCC patient subset was dichotomized at the median and tested for association with (left to right) metastatic-free survival, cancer-specific survival, and overall survival using Kaplan–Meier methodology.

Figure 4

Iron levels within the tumor microenvironment decrease with tumor progression. Tumor microenvironment iron levels (H-score) were evaluated using Prussian Blue stain of the RPCCC RCC patient TMA and tested for association with pathologic features of primary tumors including (A) tumor size (largest diameter), (B) tumor stage, (C) tumor grade, and (D) presence of sarcomatoid dedifferentiation. Representative tissue core images of tumor microenvironment iron staining are shown for low stage/grade (left) and high grade/stage (right) primary tumors of patients with (E) ccRCC and (F) pRCC. (G) Iron staining level (H-score) for the ccRCC patient subset was dichotomized at the median and tested for association with (left to right) metastatic-free survival, cancer-specific survival, and overall survival using Kaplan–Meier methodology.

Figure 5

Iron levels increase in non-neoplastic kidney during tumor progression. Normal (non-neoplastic) renal tubule epithelial iron levels (H-score) were evaluated using Prussian Blue stain of the RPCCC RCC patient TMA set and tested for association with pathologic features of primary tumors including (A) tumor size (largest diameter), (B) tumor stage, (C) tumor grade, and (D) presence of sarcomatoid dedifferentiation. Representative tissue core images of normal kidney tissues are shown at low magnification (left) and high magnification (right) for a patient with (E) low grade RCC and a patient with (F) high grade RCC. (G) Iron staining level (H-score) for the ccRCC patient subset was tested for association with (left to right) metastatic-free survival, cancer-specific survival, and overall survival using Kaplan–Meier methodology.