Advanced Intestinal Cancers often Maintain a Multi-Ancestral Architecture.

A widely accepted paradigm in the field of cancer biology is that solid tumors are uni-ancestral being derived from a single founder and its descendants. However, data have been steadily accruing that indicate early tumors in mice and humans can have a multi-ancestral origin in which an initiated primogenitor facilitates the transformation of neighboring co-genitors. We developed a new mouse model that permits the determination of clonal architecture of intestinal tumors in vivo and ex vivo, have validated this model, and then used it to assess the clonal architecture of adenomas, intramucosal carcinomas, and invasive adenocarcinomas of the intestine. The percentage of multi-ancestral tumors did not significantly change as tumors progressed from adenomas with low-grade dysplasia [40/65 (62%)], to adenomas with high-grade dysplasia [21/37 (57%)], to intramucosal carcinomas [10/23 (43%]), to invasive adenocarcinomas [13/19 (68%)], indicating that the clone arising from the primogenitor continues to coexist with clones arising from co-genitors. Moreover, neoplastic cells from distinct clones within a multi-ancestral adenocarcinoma have even been observed to simultaneously invade into the underlying musculature [2/15 (13%)]. Thus, intratumoral heterogeneity arising early in tumor formation persists throughout tumorigenesis

Apc mutations in multi-ancestral tumors.

<p>Apc mutations in multi-ancestral tumors.</p

Advanced Intestinal Cancers often Maintain a Multi-Ancestral Architecture - Fig 3

<p>In mice that were allowed to age until moribund, some tumors were adenomas with high-grade dysplasia (A and B), whereas others were intramucosal carcinomas (C-F). The neoplastic cells identified on H&E stained sections (A, C and D) were a mixture of both red and green cells (B, E and F). Panels A and B are shown at the same magnification; size bar = 1mm. Panels C and E are same magnification; size bar = 1mm. Panels D and F are 4x enlargements of the area outlined in C.</p

Multi-ancestral tumors appear to be derived from up to four founders.

<p>The expression of fluorescent proteins facilitates the use of multiphoton excitation imaging to reconstruct the tumor in three dimensions Reconstructions permit the exploration of structure beneath the surface of the tumors (A, surface; B, side view) without distortions and artifacts that occur when imaging multiple histological sections. Four distinct colored areas were evident in one tumor. Three discrete green areas were evident when the red was subtracted (C). These areas of green were separated by at least 100 microns, which is equivalent to the diameter of 1 to 2 crypts.</p

Clonal architecture of intestinal tumors from 20 mosaic mice euthanized at 150 days of age.

<p>Clonal architecture of intestinal tumors from 20 mosaic mice euthanized at 150 days of age.</p

Mosaics are a powerful experimental tool for studying tumorigenesis.

<p>The clonal architecture of colon tumors is visible <i>in vivo</i> using fluorescence endoscopy. The intestinal epithelium is a single layer of cells that is replaced every three to five days. New cells are produced from stem cells lying near the base of the each crypt. In our model, crypts are homotypic red or homotypic green with the latter being particularly evident during fluorescence endoscopy, as each crypt appears as a discrete green circle (A). Tumors can be composed of only red crypts (left panel), a mixture of red and green crypts (middle panel), or only green crypts (right panel). The tumors are outlined in white. The high variability in clonal architecture is also evident in whole mounts (B). Tumors were homotypic red (left panel), heterotypic (middle three panels), or homotypic green (right panel). The mixture of red and green crypts varies among multi-ancestral tumors, with some being primarily composed of either red crypts or green crypts, whereas others are roughly an equal number of both types. Histology is necessary to determine architecture, as tumors can appear heterotypic with neoplastic crypts of one color intermingled with normal crypts of the other color. The clonal architecture of tumors is scored from histologic sections. Neoplastic cells were visualized following H&E staining (C) with high levels of CTNNB1 (β-catenin) expression in the cytoplasm and nucleus (D, brown stain). As shown in a nearby section from the same tumor stained by immunofluorescence, neoplastic cells within a single tumor often arose from both the red and green cell lineages (E-F). Panel B photos are shown at the same magnification; size bar = 1mm. Panels C and E are shown at the same magnification; size bar = 500μm. Panel D shows a 4x enlargement in an adjacent section of the outlined area in Panel C. Panel F shows a 4x enlargement of the area outlined in Panel E. </p