Cancer from Bone Marrow. Cogle, et al
Bone Marrow Can Be A Primary Source Of Cancer
Running title: Cancer from Bone Marrow
Christopher R. Cogle1, Neil D. Theise2, DongTao Fu1, Sean Lee4, Steven M. Guthrie1, Marda L.
Jorgensen1, Doug Smith1, Swan N. Thung3, Diane Krause4*, Edward W. Scott1*
1
Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville,
FL, USA
2
Departments of Medicine and Pathology, Beth Israel Medical Center, Albert Einstein College
of Medicine, New York, NY, USA
3
4
Mount Sinai School of Medicine, New York, NY, USA
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA
* Authors contributed equally.
Correspondence:
Christopher R. Cogle, M.D., University of Florida, 1600 SW Archer Road,
ARB R4-252, P.O. Box 100277, Gainesville, FL 32610-0277, Telephone:
352-392-3058, FAX: 352-392-8530, Email: c@ufl.edu
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Cancer from Bone Marrow. Cogle, et al
SUMMARY
Bone marrow cells exhibit the ability to remodel distant organs. Here we show that marrow also
participates in the development of epithelial neoplasias of the small bowel, colon and lung. The
hematopoietic stem cell (HSC), in particular, demonstrates capability to participate in cancer
development. Furthermore, this marrow involvement in epithelial cancer does not display
fusion. Previous reports have highlighted chronic inflammation as critical to marrow
participation in cancer. Extending these findings, we found SDF-1, a powerful HSC
chemoattractant, intensely expressed in the epithelia of intestinal neoplasias containing marrowderived cells, suggesting that SDF-1 may be involved in HSC/marrow homing to cancer.
Finally, we present human data demonstrating marrow as a source of epithelial neoplasias,
underscoring the clinical relevance of these findings. Marrow participating in cancer
development may be as a direct seed or as developmental mimicry. Future endeavors will need
to distinguish between the two. Nevertheless, these findings expose new strategies for cancer
prevention and treatment.
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Cancer from Bone Marrow. Cogle, et al
INTRODUCTION
Bone marrow derived cells (BMDCs) can remodel remote organs (Alison et al., 2000; Cogle et
al., 2004; Krause et al., 2001; Theise et al., 2000). In particular, transplantation studies in
animals and humans have demonstrated that BMDCs contribute to epithelial layers of a variety
of tissues (Brittan et al., 2002; Korbling et al., 2002; Krause et al., 2001; Okamoto et al., 2002).
The origin of these epithelial cells could be from a single hematopoietic stem cell (HSC),(Krause
et al., 2001) though other contributing marrow-derived cells may also play a role in this process.
Moreover, BMDC engraftment in the gastrointestinal tract occurs by direct differentiation
without evidence of cell fusion (Harris et al., 2004). The remodeling of distant organs by bone
marrow cells occurs at very low levels in everyday physiology (Wagers et al., 2002). However,
contribution from marrow is enhanced in settings of injury or disease, which is likely related to
the homing effects of inflammation (Grant et al., 2002; Krause et al., 2001; Lagasse et al., 2000;
Theise et al., 2002; Theise et al., 2000).
A recent murine study suggested that BMDCs may contribute to cancer arising from the stomach
lining (Houghton et al., 2004). Transplantation studies performed in mice with chronic gastritis
due to bacterial infection show that resultant gastric carcinomas contained glands of marrow
origin. This study emphasizes the importance of chronic inflammation in recruiting BMDCs.
Whereas the authors purport that the recruited BMDCs then go on to serve as a source of gastric
carcinoma, another explanation for these findings is “developmental mimicry” whereby local
physical or chemical factors in the neoplastic environment influence morphologic changes in the
immigrating, multipotent BMDCs. The distinction is important. BMDCs participating as a seed
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Cancer from Bone Marrow. Cogle, et al
of epithelial cancer would clearly be a malignant process, radically changing our perspectives on
how we prevent and treat epithelial cancers. On the other hand, BMDCs participating via
developmental mimicry would initially be a benign process of recruitment and differentiation;
however, once incorporated, these multipotent developmental mimics may encourage the growth
of surrounding malignant cells with tumor growth factors, immune evasion, pro-angiogenic
factors and mobilization factors for metastasis. Developmental mimicry would also have
significant therapeutic implications, exposing new strategies to prevent and treat cancer.
Furthermore, based on in vitro cell culture in this study, it was proposed that the mesenchymal
stem cell is the cell responsible for participating in gastric carcinoma. However, this was not
tested with in vivo experimental techniques. This study also was restricted to animal studies, and
leaves to question the clinical relevance of marrow contributing to cancer.
These studies prompted us to more rigorously investigate the marrow contribution to epithelial
cancers. First, we present data from transplantation experiments in mice demonstrating that bone
marrow can contribute to spontaneously arising intestinal adenomas and carcinomas. We then
directly address the question of the marrow cell responsible for contributing to cancer by
presenting a murine lung cancer model, demonstrating that the HSC participates in cancer
growth. We also address the clinical relevance of these findings by presenting human data which
shows donor marrow contribution to secondary cancers after hematopoietic cell transplantation.
Because the chemokine, stromal derived factor 1 (SDF-1), is a potent chemoattractant of
hematopoietic stem cells and is widely expressed in many tissues during development and injury
(Aiuti et al., 1997; Butler et al., 2004; McGrath et al., 1999), we further hypothesized that SDF-
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Cancer from Bone Marrow. Cogle, et al
1 chemoattraction may be at least partly responsible for the homing and migration of BMDCs
into these neoplastic environments. Here, we show that, indeed, SDF-1 is upregulated in the
epithelial layers of intestinal neoplasias in both mice and humans.
RESULTS
Bone Marrow Contributes to Intestinal Adenomas in Mice and Humans
In order to evaluate marrow contribution to intestinal neoplasias we performed transplantation
experiments using APCmin mutant mice, which are prone to spontaneously develop adenomas
and carcinomas in the small bowel and colon. Female mice (n=4) harboring the min mutation of
the APC gene were transplanted with whole bone marrow from male APCmin mice. Three
months post-transplant, the mice were sacrificed and small bowels and colons were resected.
Adenomas and carcinomas were detected throughout the intestines of all animals. To address the
question of whether the adenomas were of host or donor origin, we utilized a combination
technique of IHC and FISH to identify neoplastic cells of donor (male) origin. Analysis of the
stained intestinal tissues demonstrated donor derived colonocytes in the adenomas and
carcinomas of all small bowel and colon specimens (Figure 1). These donor-derived adenoma
cells were identified by being cytokeratin positive, CD45RB (lymphocytes) negative, and F4/80
(granulocytes/macrophages) negative. Importantly, this triple surface protein analysis, DAPI
nuclear staining and FISH for Y chromosomes were performed on the same slide, ensuring no
false positive interpretation due to overlapping leukocyte nuclei. The evaluation of over 20,000
adenoma cells from 16 intestinal samples demonstrated that a mean 2.5% of the cells were of
bone marrow origin (range 0.2 – 12%). Furthermore, these donor-derived adenoma epithelial
cells occurred in clusters and closely approximating pockets of cytokeratin-negative donor cells
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Cancer from Bone Marrow. Cogle, et al
located in the lamina propria. To address the question of whether the donor-derived adenoma
cells represented fusion events between BMDCs and adenoma cells, we performed karyotype
analyses using confocal microscopy. None of the donor-derived adenoma cells contained a
fusion karyotype (XXY, XXXY), suggesting that fusion is an unlikely explanation (Figure 2).
At our center we also identified two women with neoplasias involving the colon after
hematopoietic cell transplantation (Table 1). The colorectal adenomas were found shortly posttransplant during colonoscopic evaluations for diarrhea. No infectious etiologies were found to
explain the diarrhea; however, graft versus host disease (GVHD) was found in surrounding
tissues, and colonic adenomas were identified and resected. Given the reports of marrow
plasticity, we questioned whether these adenomas were of host or donor origin. Because the
adenomas were found shortly after transplant, and considering the long latency period of
adenomas, it is likely that the adenomas were present in the colon before transplant and they
were expected to be entirely of host composition. Thus, we sectioned and stained the adenomas
for the presence of donor-derived cells. As expected, the neoplastic tissues demonstrated donorderived (CD45 positive, Y chromosome positive) leukocytes, which were predominantly located
in the lamina propria. To our surprise, however, the adenomas also contained donor-derived
colonocytes in the adenoma epithelial layers (Figure 1). The donor cells had lost their
hematopoietic surface protein, CD45 (leukocyte common antigen), and had adopted surface
protein expression typical of the surrounding adenoma epithelial cells (cytokeratin and mucin)
(Figure 1). Moreover, the bone marrow derived colonocytes in the adenomas were located in the
basal strata of adenoma epithelia, suggesting recent immigration. In total, over 1000 adenoma
epithelial cells were evaluated, demonstrating 1 – 4% of epithelial cells originating from donor
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Cancer from Bone Marrow. Cogle, et al
BMDCs. Information regarding the donors’ personal adenoma histories is unavailable. We
further questioned whether these marrow-derived colonic adenoma cells were a result of fusion.
In order to address this question we performed karyotype analysis. Confocal microscopy
performed to enumerate X and Y chromosomes within each donor cell nucleus showed no
evidence of a fusion sex chromosome karyotype (i.e., XXY or XXXY) (Figure 2).
Bone Marrow As a Primary Source of Skin Cancer
The observation that bone marrow incorporates into intestinal neoplasias then led us to consider
whether BMDCs can contribute to other epithelial malignancies. To evaluate other epithelial
malignancies we identified a woman who had a history of basal cell skin carcinoma 26 days prior
to hematopoietic cell transplant and 4 years after hematopoietic cell transplant from her brother
(Patient 1, Table 1). The multiparous patient was originally diagnosed with acute myelogenous
leukemia. To eradicate her leukemia she elected to undergo non-myeloablative allogeneic
hematopoietic cell transplant. Her brother donated mobilized peripheral blood cells and his
personal history of cancer is unavailable. She achieved full donor hematopoietic engraftment
three months after transplant; however had a relapse of her leukemia one year after transplant,
requiring reinduction chemotherapy and donor leukocyte infusion (DLI) from her brother. Full
hematopoietic chimerism was re-established three weeks subsequent to DLI. Post-transplant the
patient developed GVHD of the skin only. As expected, prior to transplant her basal cell skin
carcinoma was entirely female in origin (Figure 3). Although multiparous, this pre-transplant
analysis suggests that no male fetal microchimerism was evident. Her post-transplant course was
complicated by acute and chronic graft-versus-host-disease of the skin. Approximately four
years after transplant she developed another basal carcinoma involving the forehead. Biopsy of
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Cancer from Bone Marrow. Cogle, et al
the post-transplant lesion demonstrated that the epithelial malignancy was 100% male origin
(Figure 3). Furthermore, there is no evidence of fusion karyotype (i.e., XXY, XXXY) using
confocal microscopy (Figure 3).
The Hematopoietic Stem Cell Contributes to Lung Cancer in Mice and Humans
This same woman with history of skin cancer who had received a hematopoietic cell transplant
from her brother also developed lung cancer over four and one half years post-transplant (Patient
1, Table 1). She was afflicted briefly with pulmonary aspergillosis, and this was treated
definitively with antifungal therapy. Again, we questioned whether the patient’s lung cancer was
of host or donor origin. To address this question we sectioned and stained the patient’s lung
cancer with the combination techniques of immunohistochemistry for cytokeratin and FISH for
X and Y chromosomes. The lung cancer demonstrated donor origin as evidence by male cells
co-expressing cytokeratin (Figure 4). We also used confocal microscopy to perform karyotype
analysis. The donor-derived lung cancer cells demonstrated no evidence of a fusion karyotype
(i.e., XXY, XXXY) suggesting that a multipotent bone marrow derived cell from the donor
migrated to the lung and then underwent neoplastic changes (Figure 4).
Previous reports of marrow as a source of gastric cancer used in vitro testing to determine if the
hematopoietic stem cell (HSC) or the mesenchymal stem cell (MSC) is the primary source of
cancer. Given the limitations of in vitro systems, it was important for us to use an in vivo
experimental model to more rigorously examine HSC contribution to cancer. Thus, we
transplanted single, GFP tagged HSC into (primary) mice (n=120) and then after hematopoietic
chimerism was achieved (n=3) we sacrificed these mice and transplanted their bone marrow into
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Cancer from Bone Marrow. Cogle, et al
secondary recipients (n=30). All secondary recipient mice achieved GFP-hematopoietic
chimerism. Secondary recipient mice (n=9) were then injected with murine lung cancer
intramuscularly in the hind limbs. As expected, analysis of the lung cancers after 14 days of
growth demonstrated a prominent display of green intratumoral cells of HSC origin (Figure 5).
These cells were presumed to be inflammatory cells (progeny from the single green HSC).
However, staining of the lung cancer tissues for the pan-leukocyte protein CD45 only found
small pockets of donor leukocytes; the majority of these intratumoral cells were CD45 negative
(data not shown). Given the plasticity potential of the HSC, we questioned whether these HSCderived cells were contributing to the lung cancer. Thus, we evaluated the cells for evidence of
cytokeratin surface protein expression. Indeed, the tumors demonstrated HSC-derived (GFP+,
green) cells co-expressing cytokeratin (red) (Figure 5). To determine whether these doublelabeled cells (i.e., GFP and cytokeratin) were the result of fusion, karyotype analysis was
performed. All host mice and the lung cancer had a female kartyotype, allowing us to use FISH
to track the male HSC and its progeny (Figure 5). Karyotype analysis of these donor-derived,
cytokeratin positive cells found none with a fusion karyotype (i.e., XXY, XXXY) (Figure 5).
SDF-1 Expression in Neoplastic Environments of BMDC Incorporation
Based on recent rodent model findings that inflammation precedes BMDC incorporation and
eventual gastric carcinoma development (Houghton et al., 2004), we questioned whether human
BMDCs migrate to adenomas in reaction to inflammatory cues. Specifically, we hypothesized
that the powerful chemoattractant stromal derived factor 1 (SDF-1) may be at least partly
responsible for the homing and migration of bone marrow cells to the neoplastic environment
(Aiuti et al., 1997). To test this hypothesis, murine (n=4) and human (n=2) adenomas which
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Cancer from Bone Marrow. Cogle, et al
contained marrow derived adenoma epithelial cells were immunohistochemically stained for
SDF-1. Adenomas in mice and humans demonstrated intense and patchy SDF-1 expression.
Surprisingly the rich SDF-1 expression was located predominant in the epithelial layers of the
adenomas rather than in stroma or blood vessels of the lamina propria (Figure 6).
DISCUSSION
We have previously shown that the BMDCs can remodel distant organs (Cogle et al., 2004;
Krause et al., 2001; Theise et al., 2000). Whereas this BMDC incorporation is minimal in daily
physiology (Wagers et al., 2002), it becomes more apparent in settings of injury and repair,
likely reflecting the response to inflammation (Butler et al., 2005; Grant et al., 2002; Krause et
al., 2001; Lagasse et al., 2000; Theise et al., 2002; Theise et al., 2000). Indeed, recent reports
have highlighted the role of chronic inflammation in promoting marrow incorporation into
cancer (Houghton et al., 2004). Given these findings, we aimed to define the role of BMDC
participation in epithelial cancers. The present studies confirm and extend previous reports, and
is the first to raise the possibility that the HSC within the bone marrow is the responsible cell in
cancer development.
The murine transplantation studies utilizing the APCmin mutation mice provide the initial insights
into bone marrow contributing to epithelial neoplasia. Spontaneous adenomas and carcinomas in
the small bowels and colons of transplant recipient mice demonstrated adenoma cells of bone
marrow origin. Several considerations should be made. First, bone marrow incorporation into
the intestinal adenomas occurred closely approximating bone marrow derived cells located in the
lamina propria, suggesting recent immigration. Another consideration is that these BMDCs in
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the neoplasia may represent phagocytic events between the BMDC and resident adenoma cells.
If this were true then resultant cells should display hematopoietic surface proteins as well as
hyperdiploidy. However, using immunohistochemistry plus FISH, we show that these adenomas
demonstrated differentiated donor cells that expressed cytokeratin and lost expression of
hematopoietic surface proteins (CD45RB and F4/80). In addition, karyotype analysis
demonstrated no evidence for fusion (i.e., XXY, XXXY).
Given the plasticity potential of the HSC we further questioned if this particular marrow cell
participates in cancer development. Lung cancer grown in mice which were serially transplanted
from single HSC donor mice demonstrated cytokeratin positive cells of HSC origin. Our
immediate consideration was that these HSC-derived cells incorporating into lung cancer
represented phagocytosis between the HSC or its progeny and a cancer cell. To our surprise
karyotype analysis found no evidence of fusion. It has been suggested that cells which arise as
fusion products may undergo “reduction division”, dividing back into diploid cells. While that
might still be an explanation for these findings, it should be noted that in models where fusion
events have been described, most, if not all, of the fused cells persist in the tissues, without
complete “resolution” (Wang et al., 2003). Thus, direct differentiation of HSC-derived cells into
lung cancer, rather then absolutely complete and perfect resolution of every fusion event is the
most likely explanation of our current findings. These results are the first to suggest that the
HSC contributes to cancer, challenging the previous report which speculates that the
mesenchymal stem cell compartment of the bone marrow contributes to carcinomas (Houghton
et al., 2004).
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There are two possibilities to explain HSC/marrow incorporation into cancer. As a recent study
suggests, marrow derived cells may act as a direct source of cancer (Houghton et al., 2004). In
this case, BMDCs serving as a seed of cancer likely require an antecedent environment of
inflammation leading to BMDC immigration. Our study extends these findings by raising the
possibility that SDF-1 may be involved in the homing and migration of BMDCs into epithelia.
Given the potency of the SDF-1/CXCR4 axis, we report the findings that colonic neoplasias
indeed over-express SDF-1 in the epithelial layers of mouse and human adenomas, suggesting
that SDF-1 is an important signaling molecule in the recruitment of circulating marrow
stem/progenitor cells. These results also fit with the broad findings of SDF-1 in a number of
injury models (Butler et al., 2004; Hatch et al., 2002). These findings also confirm a recent
report indicating SDF-1 as a key cytokine in breast cancer growth [WEINGBERG
REFERENCE]. However, in contrast to the recent breast cancer study by Weinberg, et al, our
results found SDF-1 in the epithelial layers of intestinal adenomas and carcinomas. The
variation in location of SDF-1 expression (stromal fibroblasts versus neoplastic epithelia) may be
due to differences in sites of cancer or degree of marrow contribution to cancer. In the model
pathway of marrow as a direct source of cancer, after immigration into the inflamed epithelial
space, the multipotent BMDCs through constant exposure to inflammatory cytokines and/or
toxins (e.g., oral toxins, UV sunlight, tobacco smoking, irradiation, chemotherapy) undergo
genetic transformation events promoting self-renewal and unchecked proliferation – that is,
cancer development.
However, the previous report did not consider a second possibility that we would call
“developmental mimicry.” In this situation HSC/marrow are called into a neoplastic
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environment and then through subjection to growth factors and cell-cell contact undergo changes
in cell fate, mimicking the surrounding neoplastic cells. Given the self-renewal and high
proliferative potential of BMDCs, these marrow cells could go on to act as paracrine regulators,
providing growth factors and immune evasion for the surrounding tumor.
Whichever route most accurately describes marrow participation in cancer, both represent a
malignant process, revealing several new strategies to prevent and treat cancer.
To address the clinical relevance of these findings, we also studied cancer specimens from
patients treated at our center. The risk of developing a new cancer after blood or marrow
transplantation is estimated to be up to eight times higher than in aged-matched controls (Curtis
et al., 1997). Predisposing risk factors such as radiation, chemotherapy and use of
immunosuppressants have been recognized. However, another factor in the post-transplant
setting may be that multipotent donor marrow cells incorporate into inflamed epithelia, due to
GVHD or other inflammatory process, and then undergo pathologic changes resulting in a new
cancer. Indeed, in the human studies presented here, donor hematopoietic grafts participated in
epithelial malignancies involving the lung, colon and skin of transplant recipients. It should be
noted that preceding the neoplasias in the skin and colon specimens, GVHD was found. These
results confirm, extend and answer the clinical relevance of the previous report showing the
importance of precedent chronic inflammation (Houghton et al., 2004). One consideration is that
cancers found in the patients may have been engulfed by donor-derived bone marrow cells, such
as monocytes or macrophages. This theory of a myeloid-cancer hybrid cell has been put forth
recently by Pawelek, et al (Pawelek, 2005). These investigators studied a renal cell carcinoma
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Cancer from Bone Marrow. Cogle, et al
developing after hematopoietic cell transplantation and discovered that the tumors contained
donor DNA in addition to trisomy 17, which is a characteristic cytogenetic abnormality for this
neoplasia (Yilmaz et al., 2005). Possibilities explaining this discovery is that a cell from the
hematopoietic graft fused with a renal carcinoma creating a +Y, +17 situation or a marrow
derived cell transdifferentiated into a renal cell carcinoma. The studies presented here found no
evidence of fusion between marrow-derived cells and neoplastic cells, supporting the latter
argument. To address the question of reduction division, we scored a total of 40 Y positive
colonic adenoma cells with no evidence of hyperdiploidy. In the liver, where cell fusion has
been demonstrated in severe disease stress states, it has been postulated that 28% of donorderived hepatocytes are due to reduction division, resulting in diploid daughter cells (Wang et
al., 2003). Based on the probability of binomial distribution, the chance that we would find 40
out of 40 diploid donor-derived colonic adenoma cells amidst a background fusion resolution
rate of 28% is one in 1x1022. Thus, direct differentiation of human marrow cells, rather then
absolutely complete and perfect resolution of every fusion event, is the most likely explanation
of our current findings.
These results have strong implications for oncology, as well as stem cell biology. Our findings –
demonstrating that bone marrow cells, and specifically the HSC, incorporate into epithelial
cancers – suggest that a transplantable hematopoietic cell responds to inflammatory cues (such as
SDF-1), activates tissue-specific differentiative genetic programming and is susceptible to
further neoplastic changes. Future studies are aimed at making the important distinction between
marrow as a seed of cancer versus marrow as a developmental mimic of cancer. However, in the
meantime it appears that human BMDCs, much like their murine counterparts, may play a role in
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Cancer from Bone Marrow. Cogle, et al
epithelial cancer growth and development. With the findings presented here, new doors have
been opened to identify novel strategies for preventing and treating cancer.
EXPERIMENTAL PROCEDURES
Animals
Mice with genetic mutations in the APC gene (APCmin) and transgenic mice with ubiquitous GFP
expression (STRAIN INFO) were obtained from Jackson laboratories (Bar Harbor, Maine).
Wild-type C57BL/6 female mice were obtained from Charles River Laboratories (Wilmington,
Massachusetts). The institutional animal care and use committees of Yale University and
University of Florida approved all animal procedures.
Human Subjects
Paraffin embedded neoplastic tissues were obtained from female patients who received
hematopoietic cell transplantation from male donors, following IRB approval by the University
of Florida Health Science Center.
Murine Hematopoietic Cell Transplantation Studies
For the mouse adenoma experiments, bone marrow was harvested from a male APCmin mutant
mouse and 1x106 cells were injected intravenously into recipient female APCmin mutant mice
(n=4). To prepare recipients, APCmin female mutant mice received total body irradiation (1.1 Gy
total from a 137cesium source) followed by marrow transplantation. All recipient mice were
sacrificed 3 months post-transplant and intestines removed for fixation and staining.
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For single HSC transplants Sca-1+c-kit+Lin- HSCs were enriched by FACS sorting before
individual HSC selection with micromanipulators via fluorescent microscopy. Individual Gfp+
HSCs were then mixed with 2x105 non-Gfp+ BM cells that had been depleted of Sca-1+ cells by
magnetic beads before transplant into irradiated (0.95 Gy total, 137cesium source) hosts. For the
serial transplants, 1x103 bone marrow cells were transplanted into irradiated (0.95 Gy total)
secondary, female C57BL/6 recipients.
Mouse Adenoma Immunohistochemistry
Isotype, serum, and no primary antibody controls were included for each sample in the
immunostaining protocols. Negative and positive control tissues were processed in each staining
run. For Y FISH, CD45 and cytokeratin, 3 µm sections were deparaffinized, hydrated, incubated
in BD Biosciences Retrievagen A solution for 15 min at 100°C and then 20 min at room
temperature, and incubated in 0.2 M HCL for 12 min and 1 M NaSCN at 80°C for 20 min. Y
FISH was performed as described previously with digoxigenin-labeled Y chromosome probe and
anti-digoxigenin-rhodamine antibody (Roche Molecular Biochemicals) [NEED REFERENCE
FROM DIANE]. After Y FISH, slides were incubated in 1:20 anti- CD45RB (Santa Cruz
Biotechnology), 1:100 F4/80 (eBioscience, San Diego, CA) 1 h at room temperature, incubated
with anti-rat alexa 647 (Molecular Probes), fixed in 2% PFA in 1x PBS for 8 min, digested with
0.5 trypsin for 1 min at 37°C, washed with 5% FCS to inactivate the trypsin, incubated with
1:200 anti-pankeratin (DAKO) overnight at 4°C, incubated in 1:500 anti-rabbit-FITC (Molecular
Probes) for 1 h at 37°C, and coverslipped by using vectashield DAPI (Vector Laboratories).
Immunohistochemistry on human specimens and mouse lung cancer
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Cancer from Bone Marrow. Cogle, et al
Zinc formalin-fixed, paraffin-embedded adenoma sections were cut at 4 – 6 m and air-dried
overnight. After deparaffinization and rehydration, endogenous peroxidase activity was
quenched by application of 3% hydrogen peroxide in methanol for 10 minutes at room
temperature. Tissues to be stained for CD45 (leukocyte common antigen, LCA
DakoCytomation, Carpinteria, CA) were antigen retrieved using Trilogy unmasking solution
(Cell Marque, Hot Springs, AK). Sections stained for CK20 (cytokeratin 20, DakoCytomation,
Carpinteria, CA) were sequentially retrieved with citrate buffer (DakoCytomation,Carpinteria,
CA) and trypsin (Digest-all 2, Zymed laboratories, San Francisco CA). Endogenous biotin was
blocked with a kit (Dako, Carpinteria, CA), and primary antibody was then applied for one hour
at room temperature (1:50 for CD45 and 1:25 for CK20). Primary antibody was detected using
an LSAB2-HRP kit (DakoCytomation, Carpinteria, CA) and Diaminobenzidene (DAB).
Isotype-matched negative controls were run with each of the antibodies, finding no non-specific
binding. An appropriate positive control slide was also stained with each staining run.
SDF-1 Immunostaining
For SDF-1 staining, adenoma blocks were cut into 5 µm sections, deparaffinized and then
incubated with mouse anti–human SDF-1 Ab K15C at 1:400. Overnight incubation at 4°C was
followed by incubation with biotin-labeled rabbit anti-mouse IgG (1:100; Dako Corp.) for 30
minutes at room temperature. The sections were then incubated with ABC complex (Vector
Laboratories Inc.) and developed with DAB as substrate. They were then counterstained in
hematoxylin and covered with coverslips.
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Cancer from Bone Marrow. Cogle, et al
FISH Probing for X and Y Chromosomes
Slides were treated to two rounds of a five-minute incubation in Lugol’s solution (Sigma, St.
Louis, MO) followed by destaining in 2.5 M sodium thiocyanate. Tissue was further prepared by
incubation in 0.2 N hydrochloric acid for 30 minutes at room temperature, and incubation in 1 M
sodium thiocyanate for 30 minutes at 85 °C. Pretreatment concluded with a digestion in pepsin
at 4 mg/mL (Sigma, St. Louis, MO) in 0.9% sodium chloride, pH 2.0 for up to 60 minutes at 37
°C. Slides were next rinsed with distilled water and equilibrated in 2x saline sodium citrate
(SSC). After serial dehydration in ethanol, slides were placed on the heat plate of a Hybrite oven
(Vysis Inc, Downers Grove, IL). CEP probes for X and Y chromosomes (Vysis Inc, Downers
Grove, IL) were added to the sections and coverslips were sealed over the slides with rubber
cement. Tissue sections and probes were co-denatured at 75 °C for 6 minutes before being
hybridized overnight at 37 °C. Slides were then washed in 50% formamide in 2x SSC at 46 °C
thrice for 7 minutes each, followed by 2x SSC at 46 °C for 5 minutes, and the 4x SSC + 0.1%
Igepal (Sigma, St. Louis, MO) at 46 °C for 5 minutes. Slides were air dried in the dark and then
mounted with Vectashield containing 4,6-daminidino-2-phenylidole (DAPI) (Vector
Laboratories, Burlingame, CA).
Tissue Analysis
Slides were analyzed using a Leica laser scanning spectral confocal microscope (Leica
Microsystems, Bannockburn, IL). DAB staining for tissue specific antigens and characteristic
cellular morphology were used to specifically classify cells. Paraffin-embedded adenoma blocks
were sectioned and immunohistochemically stained with specific antibodies to identify epithelial
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Cancer from Bone Marrow. Cogle, et al
neoplastic tissues (cytokeratin) and leukocytes (CD45). Basal cell skin cancer appeared below
the epidermis. Neoplastic adenoma cells appeared elongated and large, with an epithelial
orientation and positive staining with anti-cytokeratin antisera and periodic acid Schiff (PAS)
staining. Squamous cell lung carcinoma cells were detected by their atypia, large size,
angulated nuclei, evidence of keratinzation and invasion below the basement membrane.
Leukocytes appeared small and round with positive anti-CD45 antisera staining. Y chromosome
signal was punctate, green and regularly at the nucleus perimeter. X chromosome signal was
similarly nuclear and punctate, but red.
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Cancer from Bone Marrow. Cogle, et al
Table 1. Patient Characteristics
Patient Age at
Primary
transplant Disease
(years)
1
59
AML
2
3
55
28
Transplant GVHD
2nd Cancer
N/A
PBSC
PBSC
Basal cell carcinoma
Basal cell carcinoma
Squamous cell
carcinoma of the lung
Colonic adenoma
Colonic adenoma
N/A
Yes
No
Days Post
Transplant
Percent
Donor in
Neoplasia
– 26 days
0%
1491 days
100%
1651 days
20%
AML
BM
Yes
47 days
1%
Hodgkin’s PBSC
Yes
30 days
4%
Lymphoma
GVHD, graft versus host disease; AML, acute myelogenous leukemia; PBSC, mobilized peripheral blood
stem cell; BM, bone marrow; N/A, not applicable
22
Cancer from Bone Marrow. Cogle, et al
Figure Legends
Figure 1. Intestinal neoplasias of bone marrow origin. (A – C) Intestinal adenomas and
carcinomas from the small bowels and colons of female APCmin mutant mice. (A) Micrograph of
CD45 (brown) immunostaining demonstrating few leukocytes within the adenoma (bar
represents 100 µm). (B) Micrograph of cytokeratin (brown) immunostaining displaying
adenomas with intense cytokeratin expression (bar represents 100 µm). (C) Fluorescent
micrograph of an adenoma section stained for cytokeratin (green), leukocytes (pink), nuclei
(blue) and FISH for Y chromosome (red). Arrows indicate marrow-derived cells within
adenoma epithelia. (D – F) Colonic adenomas from women who received hematopoietic cell
transplantation from male sibling donors. (D & E) Sections of the adenoma demonstrate a Y
chromosome (green) within the nucleus (blue) of a colonocyte expressing cytokeratin (brown)
and mucin (magenta). (F) Furthermore, donor-derived adenoma cells (Y chromosome, green;
nuclei, blue) were CD45 (brown) negative.
Figure 2. No evidence of fusion in bone marrow derived cells incorporated within murine
intestinal neoplasias. Intestinal adenoma spontaneously arising in a female mouse with APCmin
mutation after having received a bone marrow transplant from a male APCmin mouse. Adenomas
were stained for cytokeratin (magenta), nuclei (blue) and FISH for X (red) and Y (green)
chromosomes. Movie of serial z-steps through a murine intestinal adenoma. Arrows indicate
marrow-derived intestinal epithelial cells demonstrating no evidence for fusion (i.e., XXY,
XXXY).
23
Cancer from Bone Marrow. Cogle, et al
Figure 3. No evidence of fusion in bone marrow derived cells incorporated within human
colonic adenoma. Colonic adenoma found post-transplant in a woman who received
hematopoietic cell transplantation from her brother demonstrates adenoma cells of male donor
origin (X chromosome, red; Y chromosome, green). Movie of serial z-steps through human
intestinal adenoma. Arrows indicate marrow-derived intestinal epithelial cells incorporating
within the adenoma and displaying no evidence for fusion (i.e., XXY, XXXY).
Figure 4. Human epithelial cancers from bone marrow origin without evidence of fusion.
Basal cell skin carcinoma found pre-transplant and post-transplant in a woman who received
hematopoietic cell transplantation from her brother. (A) Fluorescent micrograph of basal cell
skin carcinoma found before transplant. As expected, skin cancer demonstrates that the entire
specimen is of female origin (nuclei, blue; X chromosome, red). (B) Four and one half years
after transplantation, skin cancer arose again and biopsy reveals that cancer is entirely of male
origin (nuclei, blue; X chromosome, red; Y chromosome, green). Confocal micrograph
demonstrates skin cancer cells with nuclei (blue) of male origin (Y chromosome, green) and no
evidence of fusion (i.e., XXY, XXXY). (C & D) Secondary lung cancer found in a woman who
received hematopoietic cell transplantation from her brother demonstrates lung cancer from
donor hematopoietic cell origin. (C) Fluorescent micrograph showing immunostaining for
cytokeratin (intense red) and FISH for Y (green) chromosomes (magnification 63X). (D)
Confocal micrograph of bone marrow derived lung cancer cells stained with cytokeratin and
FISH for X (red) and Y (green) chromosomes demonstrating no evidence of fusion. Arrows
indicate donor-derived cells.
24
Cancer from Bone Marrow. Cogle, et al
Figure 5. The hematopoietic stem cell contributes to lung cancer without evidence for
fusion. Lung cancers in female mice which received secondary transplants from a single-HSC
(GFP+,green) transplanted donor. (A) Fluorescent micrograph of lung cancer immunostained for
cytokeratin (red) (bar represents 100 µm). Lung cancers show HSC derived cells (green) coexpressing cytokeratin (red) with a merged color of yellow. Arrows indicate HSC-derived lung
cancer cells. (B) Confocal micrograph of lung cancer demonstrating non-fusion karyotype of
HSC-derived lung cancer cells. Thick sections (10 µm) of lung cancer were stained for
cytokeratin (magenta), nuclei (blue) and FISH for X (red) and Y (green) chromosomes. Arrows
indicate HSC-derived lung cancer cells expressing cytokeratin. These cells on cofocal analysis
demonstrated no evidence of fusion karyotype (i.e., XXY, XXXY).
Figure 6. SDF-1 expression in neoplastic environments. Normal and neoplastic tissues of the
intestines in mice and humans. (A & C) Normal intestines in mice and humans do not display
expression of SDF-1 (brown) in the epithelial layers. (B & D) However, adenomas in mice and
humans demonstrate intense and patchy expression of SDF-1 (brown) in the neoplastic epithelia.
25