Nitrogen isotopic composition as a gauge of tumor cell anabolism-to-catabolism ratio

Studies have suggested that cancerous tissue has a lower 15N/14N ratio than benign tissue. However, human data have been inconclusive, possibly due to constraints on experimental design. Here, we used high-sensitivity nitrogen isotope methods to assess the 15N/14N ratio of human breast, lung, and kidney cancer tissue at unprecedented spatial resolution. In lung, breast, and urothelial carcinoma, 15N/14N was negatively correlated with tumor cell density. The magnitude of 15N depletion for a given tumor cell density was consistent across different types of lung cancer, ductal in situ and invasive breast carcinoma, and urothelial carcinoma, suggesting similar elevations in the anabolism-to-catabolism ratio. However, tumor 15N depletion was higher in a more aggressive metaplastic breast carcinoma. These findings may indicate the ability of certain cancers to more effectively channel N towards growth. Our results support 15N/14N analysis as a potential tool for screening biopsies and assessing N metabolism in tumor cells.


N isotopes analysis of bulk fine needle-and micro-biopsies
As part of this study we have also investigated the δ 15 NB-C of "bulk" surgical biopsies (SI Figs. S5 and S6).We analyzed fine needle aspiration (FNA) biopsies and scalpel-taken micro-biopsies of a few hundred micrograms taken in different parts of larger frozen surgical tissue biopsies.In this case, the histopathological characterization of the different biopsies was performed in a single cryostat section per biopsy and compared to the multiple N isotopic analyses of FNA biopsies and micro-biopsies performed in different parts of the large biopsies.The purpose of this approach was to assess the heterogeneity of the large biopsies and to evaluate if it can explain the ambiguous results obtained by previous studies that performed N isotopic measurements without histopathological control of the number of tumor cells.
Surgical biopsies were obtained from the tissue bank of the Institute of Pathology of the University Hospital of Lausanne (CHUV), Switzerland (CER-VD Project ID 2020-02064).Cryostat sections for histopathological assessment were prepared as described in the main manuscript.Fine needle aspiration (FNA) biopsies and "scalpel-based" micro-biopsies were directly taken from the freshly-frozen surgical tissue biopsies stored in Tissue-Tek® O.C.T.™ compound (OCT) at -80 °C.
The tissue samples were thawed and rinsed with high-purity water before processing.For FNA biopsies, thawed tissues were sampled with a 25 G needle attached to a 10 ml syringe, the tissue biopsy was punctured, and cells and fluid were drawn up by creating a vacuum in the syringe.The sample was then transferred directly into a vial by compressing the syringe plunger and expelling the material.Scalpel-based micro-biopsies were taken by cutting off a few hundred micrograms of tissue with a scalpel.SI Tables S5 and S6 report the measurement of δ 15 N associated with the histopathological characterization of tissues from all FNA biopsies and micro-biopsies.
Micro-biopsies were sampled in 7 lung (patients 1-7) and 12 breast cancer patients (patients 6-17).FNA biopsies were performed on 7 lung cancer patients (patients 1-7) and on 9 breast cancer patients (patients 6-13 and 15).Two to three micro-biopsies and FNA biopsies per patient were measured for their nitrogen isotopic composition.δ 15 NB-C values obtained for our multiple FNA and micro-biopsies were compared with the percentage of tumor cells obtained with a microscopic assessment from a single cryostat section taken in a different part of the larger biopsy (i.e. the standard histopathological method in cancer diagnosis, SI Tables S4 and S5).
First, we assessed if tumor samples could be identified based on a statistically significant difference (p ≤ 0.05) between the δ 15 N of benign tissue and the suspected cancerous sample.Since no significant difference was observed between FNA and micro-biopsies, the measurements were combined to estimate the average and standard deviation of the suspected benign and cancerous tissue.Most of the lung cancerous tissue showed a lower mean δ 15 N value than the corresponding benign tissue (SI Fig. S5).In 6 out of 7 lung cancer biopsies the δ 15 N difference between the two tissues was statistically significant (p ≤ 0.05).In patient 5, the difference was not statistically significant (p = 0.09), due to a higher δ 15 N variability of the benign tissue; currently, we have no histopathological explanation for this variability.Results obtained from breast cancer biopsies were more variable, with higher standard deviations among analytical replicates, particularly in the benign tissues (SI Figure S6).Four cases of breast cancer, patients 14-17 (last column of SI Fig. S6), did not show the expected tendency of lower δ 15 N in cancerous tissue.Histological analysis revealed only small regions of tumor in those samples (SI Table S5), which were probably missed by the subsampling for the δ 15 N assay.Additionally, biopsies of patients 14-17 were composed of highly adipose tissue, which are not the benign tissue equivalent of the cancerous tissue and might exhibit a different δ 15 N baseline.If breast cancer patients 14-17 are excluded for the reasons described above, from the remaining 8 breast cancer biopsies 5 showed a statistically significant  S4 and S5).In general, the two approaches showed reasonable agreement.For lung cancer, in 5 out of 7 cases, the microscopic estimate fit within the propagated uncertainty of the δ 15 N-based estimate.For breast cancer, 9 out of 12 cases fit.Nevertheless, differences between these two approaches for estimating tumor percentage are to be expected.The tumor distribution of the surgical biopsies likely varied importantly with depth, and the visual characterization only captures a tumor cell density on a single section through the biopsy.In addition, in breast tissue samples, the variable occurrence of adipose tissue likely added to the isotopic variation.Thus, we suggest that the δ 15 Nbased estimate, which is based on the analysis of multiple measurements, may provide a better approximation of the average TCD in the biopsy.In any case, the large variability observed in our measurements across different patients and cancer types illustrates the importance of taking into account tissue heterogeneity when interpreting isotopic results.In this sense, our results illustrate the challenges and limitations of N isotopic measurements performed without histopathological control of the benign and cancerous tissue analyzed.This provides an explanation for the ambiguous results obtained in previous studies that measured N isotopic differences in "bulk" human biopsies without precise histopathological control of the sample analyzed.SI Tables S1-S5: All tables include patient number, tissue type, gender and age, δ 15 N (1.s.d.), tumor cell percentage as quantified by pathologist, pathological description of cryostat section, microscopic images of cryostat sections.S1 Lung

SI Table
Breast cryostat sections of patients 1 to 5. In panels a) and b) top part is cancerous tissue, bottom part is benign tissue, sampled on separate cryostat sections.In patients 3 to 4, i.e. panels c to e, one single cryostat section containing both, cancerous and benign tissue was sampled.The δ 15 N (‰ vs. air) of each cryostat sections is indicated in red writing for cancerous sections/areas and in black for benign sections/areas.On the right of each panel, the average δ 15 N (‰ vs. air) of all sections is indicated.4 SI Fig. S3: Kidney cryostat sections of patients 1 to 4, top of each panel is cancerous tissue, bottom of each panel is benign tissue.The δ 15 N (‰ vs. air) of each cryostat sections is indicated in red writing for cancerous sections and in black for benign sections.On the right of each panel, the average δ 15 N (‰ vs. air) of all sections is indicated.SI Fig. S4: Kidney cryostat sections of patients 5 to 8, top of each panel is cancerous tissue, bottom of each panel is benign tissue.The δ 15 N (‰ vs. air) of each cryostat sections is indicated in red writing for cancerous sections and in black for benign sections.On the right of each panel, the average δ 15 N (‰ vs. air) of all sections is indicated.