Disseminated Tumor Cells Persist in the Bone Marrow of ...Tumor and Stem Cell Biology Disseminated...

Tumor and Stem Cell Biology

Disseminated Tumor Cells Persist in the BoneMarrow of Breast Cancer Patients throughSustained Activation of the Unfolded ProteinResponseKai Bartkowiak1, Marcel Kwiatkowski2, Friedrich Buck2, Tobias M. Gorges1,Lars Nilse3, Volker Assmann1, Antje Andreas1, Volkmar M€uller4, Harriet Wikman1,Sabine Riethdorf1, Hartmut Schl€uter2, and Klaus Pantel1

Abstract

Disseminated tumor cells (DTC), which share mesenchymaland epithelial properties, are considered to be metastasis-initiat-ing cells in breast cancer. However, the mechanisms supportingDTC survival are poorly understood. DTC extravasation into thebone marrow may be encouraged by low oxygen concentrationsthat trigger metabolic and molecular alterations contributing toDTC survival. Here, we investigated how the unfolded proteinresponse (UPR), an important cytoprotective program induced byhypoxia, affects the behavior of stressed cancer cells.DTC cell linesestablished from the bone marrow of patients with breast cancer(BC-M1), lung cancer, (LC-M1), and prostate cancer (PC-E1)weresubjected to hypoxic and hypoglycemic conditions. BC-M1 andLC-M1exhibitingmesenchymal and epithelial properties adaptedreadily to hypoxia and glucose starvation. Upregulation of UPR

proteins, such as the glucose-regulated protein Grp78, inducedthe formation of filamentous networks, resulting in prolifer-ative advantages and sustained survival under total glucosedeprivation. High Grp78 expression correlated with mesenchy-mal attributes of breast and lung cancer cells and with poordifferentiation in clinical samples of primary breast and lungcarcinomas. In DTCs isolated from bone marrow specimensfrom breast cancer patients, Grp78-positive stress granuleswere observed, consistent with the likelihood these cells wereexposed to acute cell stress. Overall, our findings provide thefirst evidence that the UPR is activated in DTC in the bonemarrow from cancer patients, warranting further study of thiscell stress pathway as a predictive biomarker for recurrentmetastatic disease. Cancer Res; 75(24); 5367–77. �2015 AACR.

IntroductionAfter infiltration of secondary sites, adaptation of disseminated

tumor cells (DTC) to novel microenvironmental conditions is arelevant prerequisite for metastases formation. Therefore, theunderstanding of DTC survival strategies in response to externalstimuli can provide important insights into the mechanisms ofearly stages of cancer metastasis. Among other organs, the bonemarrow, seems to be an interesting reservoir for DTCs in breast,lung, and prostate cancer (1). In the bone marrow, an oxygenpartial pressure of only approximately 1% O2 (hypoxia) wasdetected in the human hematopoietic stem cell niche (2). Exper-

imental studies indicate that DTCs might settle in this niche (3)and that in the extravascular region surrounding the sinusoidseven lower O2 concentrations are present than in hematopoieticstem cell niche (4).

In human breast cancer, DTCs frequently extravasate into thebone marrow, survive chemotherapy, and predict the clinicaloutcome of patients (5–7). Within the circulating tumor cell(CTC) and DTC pool, tumor cells with mesenchymal attributesmight play an important role in the metastatic process, becausethese cells have stem cell properties and they are more resistant tochemotherapies (8).

Tumor hypoxia has attracted interest, as hypoxia is not onlyimplicated in theWarburg effect, but can also promote tumor celldissemination or DNA damage (9, 10). Furthermore, it has beenshown in amousemodel that the capability ofDTC to settle downin the hematopoietic stem cell niche is mediated by the C-X-Cchemokine receptor type 4 (CXCR4; ref. 3). Hence, a significantDTC subpopulation may be subjected to low oxygen concentra-tions in bone marrow (11, 12).

When tumor cells are confronted with hypoxic stress, cytopro-tective programs are activated. In this context, the unfoldedprotein response (UPR) is of particular interest. We have previ-ously identified a strong UPR-positive DTC subpopulation in thebone marrow sharing mesenchymal attributes with low cytoker-atin expression (11). Under hypoxic conditions, proteins in theendoplasmic reticulum (ER) canbecomemalfolded, in a so-called

1Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 2Institute of Clinical Chemistry, Uni-versity Medical Center Hamburg-Eppendorf, Hamburg, Germany.3Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-Universit€at Freiburg, Freiburg,Germany. 4Department of Gynecology,University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Klaus Pantel, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Phone: 4940-7410-53503; Fax: 4940-7410-55374; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-3728

�2015 American Association for Cancer Research.

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ER stress (13). If ER stress cannot be stopped, irreversible pertur-bation of the ER function leads to cell death. Activation of theUPR chaperones and oxidoreductases is a rescue program thatrefolds malfolded proteins when the perturbation is resolvableunder mild stress. Besides resistance against cell stress, the UPRchaperone 78 kDa glucose-regulated protein (Grp78) is able tomediate resistance against cytotoxic chemotherapy like epirubicin(14). In prostate cancer, the Grp78 levels correlate with thereceptor tyrosine kinase ErbB-2 level and high Grp78 expressionin the primary tumor is linked to poor survival (15, 16). We havepreviously observed that ErbB-2 is frequently expressed onDTCs in the bone marrow of breast cancer patients (17) andthat Grp78 expression is induced after ErbB-2 stimulation withgrowth factors (11).

Here, we were interested in stress responses of cancer cells,when they are subjected to adverse microenvironmental condi-tions as found in the in bone marrow.

Materials and MethodsSee Supplementary Data for extended description of the Mate-

rials and Methods.

PatientsThe human investigations were performed according to the

Helsinki rules after approval was obtained by the ethics commit-tee of the Medical Association Hamburg. From all patients,written informed consent was obtained prior to any study-relatedprocedures. Samples from women with breast cancer or healthycontrol persons treated at the University Medical Center Ham-burg-Eppendorf (Hamburg, Germany) were used. The patientsunderwent bonemarrow aspiration at the time of primary surgery(DTCanalyses). ForCTC analyses, bloodwas drawn frompatientswithmetastatic breast cancer after resection of the primary tumor.Patients with a known positive CTC/DTC status were selected.

DTC/CTC detectionFor DTC detection the pan-cytokeratin antibody AE1/AE3

(Monosan) was applied. The primary antibody against Grp78(rabbitmonoclonal,C50B12,Cell SignalingTechnology)wasused.For CTC detection, the pan-cytokeratin antibody KL1 (Abcam)wasapplied. Immunocytochemical double staining was performedapplying the anti-G6PD antibody (rabbit polyclonal, Abcam).

Immunohistochemical staining of organ metastasesParaffin-embedded organ metastases on microscope slides of

breast cancer patients were applied for Grp78 analysis. Thesecondary antibody (polyclonal rabbit anti-mouse immunoglo-bulins; DAKO) was applied in a 1:20 dilution for 30minutes. Forthe alkaline phosphatase - anti-alkaline phosphatase procedure,the mouse monoclonal antibody (DAKO) was diluted 1:100 andincubated for 30 minutes. The Grp78 antibody was used in a1:200 dilution.

Grp78 tissue microarraysGrp78 expression in human tumor samples was assessed by

immunohistochemical staining. A breast cancer tissue microarray(TMA, see ref. 18) anda sector of a lung cancer TMA(19)wereusedfor the Grp78 analysis. One-hundred and eighty-two primarybreast and36primary lung tumors became eligible for evaluation.For each tissue sample, the staining was estimated on a three-step

scale (0, 1, and 2) based on staining intensity and percentage ofstained cells. The criteria for these groups were as follows: 0: weak(close to detection limit or no staining); 1: moderate (1þ stainingin�50% of cells or 2þ staining in �10% of cells); 2: strong: (1þstaining in >50% of cells or 2þ staining in >10%). The statisticalanalyses were performed using the Pearson c2 or the Fisher exacttest. P values lower than 0.05 were considered statisticallysignificant.

Cell lines and culture conditionsAll cell lines were cultured at 37�C in a humidified environ-

ment. The DTC cell lines BC-M1, LC-M1, and PC-E1 (20) wereessentially cultured as described (21). The DTC cell lines werecultured with 5% CO2 and 10% of oxygen. MCF-7 (from ATCC),Hs578t (from Thomas Dittmar, University of Witten/Herdecke,Germany) and MDA-MB-231 and MDA-MB-468 (both from cellLines Service, Eppelheim, Germany) were cultivated in DMEMwith 10% fetal calf serum and 2 mmol/L L-glutamine. MDA-MB-468 cells that overexpress ErbB-2 (MDA-468 ErbB-2; MDA-468PM), and the corresponding G418-resistant control cells werecultivated as described (22). Cell lines cultured in DMEM werekept in presence of 10% CO2 and the remaining gas mixture wasatmospheric air. These cell culture conditions refer as to "standardcell culture condition" in this work. Cell lines were used within 6months after resuscitation; authentication method by the cellbanks: STR/gDNA.

Cultivation of the cell lines in presence of 1% O2

Cultivation of the cell lines in presence of 1% of O2 (hypoxia)was performed using the incubator Heracell 15 (Thermo FisherScientific). The oxygen partial pressure was adjusted by N2, andthe other cell culture conditions were the same as for the standardcell culture conditions.

Cultivation of the cell lines under glucose starvation conditionsWhen the cell lines were cultured in medium that contains no

glucose (Glu0), the other culture conditions were the same as forstandard culture conditions. ATP was purchased from Sigma-Aldrich. The final concentration was 2.5 mmol/L.

SDS-PAGE, Western blot, and densitometric analysisWestern blot analyses were essentially performed as described

(11). Experimental details, including the applied antibodies, arespecified in supporting information.

ResultsCharacterization of DTC with mesenchymal attributes

As breast, lung, and prostate cancers show a high incidence ofbone metastases, we compiled bone marrow DTC cell linesgenerated from DTC of cancer patients with these tumor entities:BC-M1 (breast cancer), LC-M1 (lung cancer), and PC-E1 (prostatecancer; refs. 11, 20, 23–25). BC-M1, LC-M1, and PC-E1 expressCXCR4 that may principally enable these cells to harbor in thehematopoietic stem cell niche (Fig. 1A). These cell lines exhibitmesenchymal attributes like vimentin expression but retain someepithelial attributes like expression of cytokeratins (Fig. 1A andSupplementary Fig. S1A; ref. 11).

Compared with MDA-MB-468 (MDA-468) and MCF-7 cellsthat exhibit a high degree of epithelial differentiation BC-M1,LC-M1, and PC-E1 showed a very high level of the UPR proteins

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(Fig. 1B and C). Besides Grp78, proper folding of ER residentproteins is accompanied by the UPR chaperones 94 kDa glucose-regulated protein (Grp94) and hypoxia upregulated protein 1(Hyou1) and by the oxidoreductase protein disulfide-isomerase(PDI). ErbB-2 is present in BC-M1 and LC-M1 cells at comparablelevels withMDA-MB-231 (MDA-231) and Hs578t (Fig. 1B and C).

To analyze how the cell lines respond to oxygen-limitingconditions as found in the bone marrow, we subjected the celllines to hypoxia (1% O2) and compared the growth rates of thecell lines by counting of the viable cells (Fig. 1D). All analyzed celllines proliferated under hypoxia. Standard cell culture conditions

(designated with "S", see Fig. 5C for additional values) served asgeneral reference system. Using these values, the growth rates ofthe cells under hypoxia (Fig. 1D) and glucose starvation (seebelow) can be comparedwith the growth rates under standard cellculture conditions.

Divergent development of tumor cells under hypoxicconditions

We evaluated a potential upregulation of Grp78 and PDI inresponse to hypoxia (Fig. 2A and B). Instead of the expectedinduction, Grp78 was downregulated in MDA-231, MCF-7, and

Figure 1.Comparison of the DTC cell lines BC-M1, LC-M1, and PC-E1 with breast cancer cell lines with different degrees of epithelial differentiation. A, determinationof the degree of epithelial differentiation. B, expression of the unfolded protein response proteins and EGFR, ErbB-2. C, quantitative Western blot analysis ofGrp78 and PDI. D, growth rates of breast cancer cell lines and DTC cell lines under hypoxia (1% O2). At time points designated with "S," cells were cultivatedunder standard cell culture conditions. Error bars, SD from the mean value.

DTC Adaptation Strategies to Cell Stress

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LC-M1. Only in Hs578t Grp78 was induced. All investigated celllines induced EGFRwith the only exception of Hs578t. Moreover,stabilization of HIF1a by cobalt chloride (26) resulted in induc-tion of Grp78 expression, suggesting an interaction of HIF1a toGrp78 (Fig. 2C).

We assumed that induction of EGFR expression (probably withconcomitant minor changes in ErbB-2 expression) may lead tomitigation of the Grp78 expression under hypoxia.

Therefore, we tested if EGFR/ErbB-2 expression affects theGrp78 and PDI induction under hypoxia (Fig. 2D; see alsoSupplementary Fig. S1B–S1D). ErbB-2 was stably overexpressedin MDA-468 cells and the resulting cell line was named MDA-468 ErbB-2. MDA-468 PM cells expresses ErbB-2, in whichtyrosine 1248 was replaced by phenylalanine (24), in which

neither Grp78 nor PDI expression was induced after growthfactor stimulation (11) and served as a negative control. MDA-468 control carries an expression vector without insert. Underhypoxia, Grp78 induction was more pronounced in MDA-468ErbB-2 than in MDA-468 control (Fig. 2D). Conversely, PDIwas stronger induced in MDA-468 control than in MDA-468ErbB-2. In MDA-468 PM neither Grp78 nor PDI were markedlyinduced.

A second faint Grp78 signal was detected in BC-M1 and MDA-231 (Supplementary Fig. S1E). To confirm the assumption thatthis signal was a cytoplasmic splice variant of Grp78, Grp78va(27), we performed RT-PCR analyses (Supplementary Fig. S1Fand S1G). ThemRNA of Grp78va is detectable in the cell lines forat least 75 hours under hypoxia.

Figure 2.Response of the analyzed cell lines to hypoxia (1% of O2) by Western blot analysis (A and B) and cobalt-induced stabilization of HIF1a (C). D, ErbB-2–dependentprotein induction (Grp78) and attenuation (PDI) under hypoxia and quantitative Western blot analysis of Grp78 and PDI. MDA-MB-468 (MDA-468) ErbB2carries an ErbB2 expression vector; MDA-468 PM expresses ErbB-2, inwhich Y1248 of the ErbB-2 proteinwas replaced by phenylalanine; MDA-468 control carries anexpression vector without insert. Error bars indicate the SD from the mean value. Time points 0 hours signify standard cell culture conditions.

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Detection of an acute stress response in cell lines andDTC ex vivo

We observed the formation of large Grp78-positive aggre-gates after subjection of the cell lines to hypoxic conditions(Fig. 3A). In MDA-468 ErbB-2 Grp78/ErbB-2 cytoplasmic colo-calization in these granules was observed. Under standard cellculture conditions the ER protein Grp78 showed an evencytoplasmic distribution. When the cells were subjected tohypoxic conditions, the granule formation reached a maximumat 75 hours of 1% O2 and after 150 hours the distributionof Grp78 resembled again those of the standard cell culturecondition. Grp78-positive granules were also observed in MDA-468 control, MDA-468 PM and MCF-7, whereas in Hs578t andMDA-231 only little formation of the granules was detected. InBC-M1 (Fig. 3B) and LC-M1, the formation of Grp78-positivegranules was not observed.

To validate our experimental findings, we analyzed DTC in thebone marrow of breast cancer patients using density gradient–based enrichment of DTC and cytokeratin as marker for DTCssimilar to our previous work demonstrating the prognostic rele-

vance of DTC in breast cancer patients (5, 28). Using cytokeratin/Grp78 double immunostaining, we detected cytokeratin/Grp78-positive DTCs that showed large cytoplasmic Grp78 granulessimilar to those found in our cell lines after cultivation of thecells under hypoxia (Fig. 3C). Similar to Grp78, we found PDI-positive granules in DTCs (Fig. 3D). About each fifth DTC waspositive for the granules of these two UPR-proteins, whereas forthe majority of DTCs no granules were observed.

Protein expression analysis of CTC/DTC with mesenchymalattributes

Triple-negative primary breast tumors frequently release tumorcells with mesenchymal attributes into the blood (29). We mod-eled the in vivo situation by analysis of the DTC cell line BC-M1and the triple-negative cell line MDA-468. The relative proteinabundance in MDA-468 and BC-M1 were determined by massspectrometry applying metabolic labeling (stable isotopic label-ing of amino acids in cell culture, SILAC). Quality testing (Sup-plementary Fig. S2) showed that samples are of suitable qualityfor mass spectrometric analysis.

Figure 3.Immunocytochemical analysis of MDA-468 ErbB-2 and BC-M1 for Grp78 and ErbB-2 subcellular location after cultivation of the cells under 1% of O2 (A and B).Scale bar, 20 mm for all images. C and D, detection of large cytoplasmic granules of Grp78 (C) and PDI (D) in DTCs from the bone marrow of breast cancerpatients by immunocytochemical double staining. Time point 0 hours, standard cell culture conditions.






Detected proteins relevant in tumor cell dissemination arepresented in Fig. 4; Supplementary Tables S1–S3. For seven ofthe proteins, confirmation by Western blot analysis is shownin Fig. 4B (quantitative analyses in Supplementary Fig. S3A).These results suggested a potential contribution of the induciblenitric oxide synthase (NOS2) in DTCs. Isoform 2 of voltagedependent anion selective channel protein (VDAC2; Fig. 4) isinvolved in the generation of nitric oxide. In breast cancer, NOS2

can produce nitric oxide, and on mRNA level we found aninduction of nos2 in BC-M1 andLC-M1 (Supplementary Fig. S3B).

Several enzymes of the pentose phosphate pathway (PPP) werepresent in BC-M1 at lower level comparedwithMDA-468 (Fig. 4),whereas glycolytic proteins showed similar values. For the PPPenzymeGlucose-6-phosphate 1-dehydrogenase (G6PD), an asso-ciation with the tumor cell phenotype was observed (Fig. 1and Fig. 4B). Very high G6PD expression was observed in cells

Figure 4.A, comparison of the relative protein levels in BC-M1 with MDA-MB-468 (MDA-468) by SILAC LC/MS-MS analysis. The average ratios of the peptide signalintensities for the individual proteins are shown. A positive value of the average ratio indicates an increased protein expression in BC-M1 and a negative valuesignifies an increased protein expression in MDA-468. Error bars, the SD from the average value. For details, see Supplementary Table S1. B, confirmation of theprotein levels by Western blot analysis for the denoted proteins (quantitative analyses, Supplementary Fig. S3). C, analysis of the cellular responseafter Grp78 knock down in BC-M1 by Western blot analysis.

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with a high degree of epithelial differentiation like MDA-468compared with cells that share epithelial/mesenchymal attributeslike MDA-231. DTC cell lines like BC-M1 showed even lowerG6PD expression compared with MDA-231. Grp78 knockdownin BC-M1 (Fig. 4C) induced the expression of G6PD, whereas for

6PGDadownregulationwas observed.G6PD,which catalyzes therate-limiting step in the PPP, was analyzed in CTCs of breastcancer patients (Fig. 5A). Cytokeratin-positive CTCs that weredetected in the blood of breast cancer patients uniformly revealeda lowG6PD signal intensity. For a comparison of theG6PD levels,

Figure 5.A, analysis of circulating tumor cells(CTC) from the blood of breastcancer patients for G6PD byimmunocytochemical double staining.B–D, response of the denoted cell linesto glucose withdrawal. S, standardcultivation with glucose; Glu0,glucose-free RPMI medium. B,morphologic analysis of the cells bybright field microscopy. C,proliferation rates; P value (Studentt test). D, Western blot analysis aftercultivation under glucose shortagecompared with standard cell culturecondition. MDA-231 cells thatdetached from the cell culture flaskafter glucose withdrawal wereanalyzed separately. Error bars, SDfrom the mean value. Scale bars,50 mm (A) and 200 mm (B).






BC-M1, MDA-231, and MDA-468 were spiked into the blood orbone marrow of healthy control persons (Supplementary Fig.S3C–S3E). The cytokeratin-positive/G6PD weakly positive CTCphenotype in cancer patients looks similar to the MDA-231phenotype in vitro.

DTC adaptation strategies to glucose starvationWe investigated a cellular response of tumor cells with mes-

enchymal properties when they are cut off from glucose supply(Glu0) by culturing the cells in glucose-freemedium (Fig. 5B). TheDTC cell lines with a pronouncedUPRphenotype BC-M1 and LC-M1 induced a filamentous network upon glucose starvation. The

cell lines PC-E1, MDA-231, and Hs578t that exhibit a reducedlevel of the UPR proteins did not form such structures. We wouldlike to call these cell structures "sprites" analogous to the fila-mentous structures of a subdivision of lightings. All cells grewslower under glucose starvation compared with standard cellculture conditions. BC-M1 and LC-M1 proliferated under glucosestarvation, whereas for PC-E1 and MDA-231 reduced cell num-bers were observed (Fig. 5C). TheWestern blot analyses revealed amassive induction of the synthesis of UPR proteins Grp78 andGrp94 and downregulation of HIF1a (Fig. 5D). BC-M1 cells thatwere subjected to glucose shortage were stained for Grp78 (Fig.6A). These analyses confirmed that the sprites are Grp78 positive.

Figure 6.A, Grp78 detection in BC-M1 by immunofluorescence after glucose starvation for 130 hours. B, response of the DTC cell lines BC-M1 and LC-M1 to ATP treatmentand to Grp78 knockdown (BC-M1). S, standard cultivation; Glu0, glucose-free medium. For Glu0 þ ATP (150 hours), the cells were incubated right from thestartwithATP. ForGlu0 (150hours)þATP (48hours), the cellswere first cultivatedwithout glucose for 150 hours followedby addition ofATP in glucose-freemediumfor 48 hours (P value, Student t test. C, Grp78 protein expression on samples of a primary breast tumor tissue microarray (TMA) from 182 cases. D,immunohistochemical Grp78 detection in organ metastases of breast cancer patients. All samples were from different patients and the matched primary tumorswere not available. Scale bars, A and B, 200 mm; C, 100 mm; D, 50 mm. Error bars, SD from the mean value.

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As glucose is the major ATP source in cancer cells, we investi-gated whether the filaments in the DTC cell lines may be reversedby external ATP supply (Fig. 6B). Supplementation of the Glu0

mediumwith 2.5mmol/L ATP suppressed thefilament formationa priori. Cultivation of the cells for 150 hours under Glu0 followedby Glu0 medium supplemented with ATP for 48 hours reversedthe filament formation. Grp78 knockdown suppressed the fila-ment formation in BC-M1 (Fig. 6B), suggesting a contribution ofGrp78 to the sprite formation.

Grp78 expression in primary and metastatic lesions fromcancer patients

Grp78 was selected as an example of the UPR proteins for theanalysis in primary breast tumors. The Grp78 staining results areshown inFig. 6Cand thecorrelationof theGrp78 staining intensitywith clinicopathologic parameters are presented in Table 1. Intotal,Grp78staining resultswereobtained for182primary tumors:21 cases (11%) were Grp78 negative, 35 cases (19%) showedmoderate staining, and 126 tumors (69%) expressed high levels ofGrp78. Grp78 staining intensity was correlated to the tumordifferentiation grade (P < 0.0001); poorly differentiated tumorswere more often strongly Grp78-positive than well-differentiatedtumors. The correlation of the Grp78 staining intensity with thetumor differentiation grade was also observed on primary tumorsamples from lung cancer (n ¼ 36; P ¼ 0.024).

The presence of Grp78 in organ metastases was confirmed onsamples from breast cancer patients (Fig. 6D). Even though theGrp78 signals in the bonemetastases was very heterogeneous, theoverall signals were much stronger than for the liver metastases.

Analysis of tumor cell spread via lymphatic vessels showedGrp78 expression in 50 of 53 lymph node metastases. Threespecimens showed no Grp78 signals. From 27 patients, matchedpairs of the primary tumor and lymph node metastases wereavailable (similar Grp78-staining: 19 cases, reduced or elevatedstaining in the lymph node metastases 5 or 3 cases, respectively).

DiscussionWe showed that DTCs express high levels of UPR proteins like

Grp78 and form cytoplasmic Grp78-positive granules when sub-jected to acute hypoxic stress. These findings on cell lines werevalidated on clinical samples from cancer patients. Under acutehypoxic stress, the mRNA translation is aborted, whereas mRNAsthat contain internal ribosome entry sites (e.g., grp78; ref. 30) canbe translated by the cap-independent translation leading to novelsynthesis of these proteins. Ribosomes with stalled protein trans-lation disassemble and the 40S ribosomal subunits accumulate tocytoplasmic foci with a diameter of 0.1 to 2 mm, which can bedetected by microscopic analysis. The large foci are called stressgranules, and foci with a lower diameter are called processing

Table 1. Association of Grp78 expression with clinicopathologic properties on a breast cancer tissue microarray

Grp78 weak(score 0)

Grp78 moderate(score 1)

Grp78 strong(score 2)

n (%) n (%) n (%) Pa

Primary tumorsAll 21 (12) 35 (19) 126 (69) —

Histology 0.967Infiltrating ductal 14 (67) 25 (71) 94 (75)Infiltrating lobular 5 (24) 5 (14) 19 (15)others 2 (10) 5 (14) 12 (10)

DTC status (bone marrow) 0.262Positive 8 (38) 11 (31) 28 (23)Negative 13 (62) 24 (69) 94 (77)

Lymphangiosis 0.117Positive 0 (0) 1 (3) 14 (14)Negative 14 (100) 28 (97) 89 (86)

Stage 0.348T1 10 (48) 24 (69) 61 (49)T2 8 (38) 10 (29) 54 (43)T3 1 (5) 1 (3) 3 (2)T4 2 (10) 0 (0) 7 (6)

Lymph node status 0.810Positive 9 (43) 12 (34) 46 (37)Negative 12 (57) 23 (66) 79 (63)

Grading <0.0001G1 4 (19) 0 (0) 1 (1)G2 12 (57) 27 (79) 59 (49)G3 5 (27) 7 (21) 61 (50)

Hormone receptor status (ER þ PR) 0.197Positive 19 (90) 30 (86) 96 (76)Negative 2 (10) 5 (14) 30 (24)

ErbB-2 status 0.606Positive 0 (0) 1 (3) 5 (5)Negative 18 (100) 34 (97) 105 (95)

Subtyping 0.181ER þ PR positive 16 (76) 29 (83) 79 (63)Triple negative 2 (10) 5 (14) 25 (20)ErbB2 positive 0 (0) 1 (3) 5 (4)n.a. 3 (14) 0 (0) 17 (14)

aPearson c2 or Fisher exact test.






bodies or GW bodies (31). In principle, these observations arecompatible with a hectic stress phase in which the cells reorganizetheir proteome to cope with the new microenvironmentalconditions.

Moreover, we have found that these granules recede after 150hours of hypoxia in cancer cells in vitro. We assume that thesegranules are preferentially detectable in susceptible DTCsexperiencing acute stress, as stress granules are rapidly inducedin response to environmental stress like hypoxia (32). This wouldbe the case, when such DTCs just have settled in hypoxic areas ofthe bone marrow. Because of this transient nature of stressgranules, it might be assumed that stress granules may be appli-cable for the detection of a subpopulation of freshly (�150hours)settled DTCs. DTCs that already have undergone the stress adap-tation prior to sampling, intrinsic stress tolerant DTCs, as well asDTCs that are unable to undergo stress adaptation would appearas stress granule-negativeDTC (11).Despite of the contribution ofGrp78 to stress granules (33), it has to be emphasized that theUPR and stress granules essentially belong to different cytopro-tective programs and intervene on different steps of the proteinsynthesis (13, 31).

Beyond hypoxia, alterations in the glucose supply may affectthe phenotype of bone marrow DTCs, because we found thattumor cells withmesenchymal attributes exhibit strongly reducedlevels of the PPP enzyme G6PD. To get deeper insights intoresponses of DTC to glucose supply, we cultured the cells inglucose-free medium so that the sole nutrients were the aminoacids from the culturemedium. Apart fromdirect oxidation of theamino acids for energy conversion, it is conceivable that tumorcells are able to use the amino acids for subsequent biochemicalreactions similar to the reverse reaction of the glycolysis, thegluconeogenesis (34, 35). Under these conditions, the cells mightbe able to undergo a limited oxidative phosphorylation. Ourobserved response of BC-M1andLC-M1 to glucose deprivationbythe sprite formation may be regarded as a strategy to cope withnutrient deficiency afterDTC colonization of the bonemarrow. Astumor cells withmesenchymal attributes are less addicted to cell–cell contacts, these cells might establish more readily a filamen-tous network with only a low amount of physical contact to theirneighbor cells.

We detected in BC-M1 cells large amounts of fibronectin bymass spectrometry, which may provide additional informationabout the nature of the sprites. Structures that look similar tothe sprites were observed before and are strongly positive forextracellular marker proteins like fibronectin, but negative forendothelial cell markers (36). In breast cancer, such structureswere observed before (37, 38), but were not implicated as anadaptation strategy of DTCs. These structures are the resultof vasculogenic mimicry. Vasculogenic mimicry is a primitivevariety of tumor angiogenesis in which poorly differentiatedtumor cells form tube-like structures that provide a simplesecondary circulation system independent from angiogenesis(39). The vasculogenic mimicry patterns do not develop frompreexisting blood vessels, but from tumor cells. Vasculogenicmimicry can be induced by ischemia or hypoxia and is

regarded as a main source of blood supply in tumor growthprior to angiogenesis (40). Because of their plasticity, cancerstem cells (40) and cells that passed epithelial-to-mesenchy-mal transition (41) are implicated in the vasculogenic mimicryformation in tumors.

The heterogeneous Grp78 expression in bonemetastasesmightdepict the versatile Grp78 regulation depending on intrinsic andextrinsic stimuli like the degree ofmesenchymal attributes, EGFR/ErbB-2 expression, hypoxia, glucose starvation, or a combinationof those factors. In addition, the oxygen concentration in thenormal bone can vary on even low micrometer scale (4, 42). It isconceivable that similar variations in the microenvironmentalconditions might also occur in larger tumor cell colonies likemicrometastases leading to heterogeneous Grp78 induction onsmall spatial areas.

Grp78 in CTCs/DTCs might be explored in the future as anew risk predictor of metastatic relapse. The potential contri-bution of EGFR to Grp78 downregulation under hypoxia mightbe a potential way to influence Grp78 protein levels in CTCsand DTCs.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K. Bartkowiak, H. Schluter, K. PantelDevelopment of methodology: K. Bartkowiak, M. Kwiatkowski, A. Andreas,H. Schluter, K. PantelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Bartkowiak, M. Kwiatkowski, F. Buck, T.M. Gorges,A. Andreas, V. Muller, S. Riethdorf, H. Schluter, K. PantelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Bartkowiak, M. Kwiatkowski, F. Buck, L. Nilse,V. Muller, H. Wikman, S. Riethdorf, H. Schluter, K. PantelWriting, review, and/or revision of the manuscript: K. Bartkowiak, M. Kwiat-kowski, F. Buck, T.M. Gorges, V. Muller, H. Wikman, S. Riethdorf, H. Schluter,K. PantelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Bartkowiak, M. KwiatkowskiStudy supervision: K. PantelOther (experimental conduct: SILAC and mass spectrometry): K. Bartkowiak

AcknowledgmentsThe authors thankMalgorzata Stoupiec for excellent technical assistance and

Alexander Schulze for the CXCR4 antibody.

Grant SupportThisworkwas supported by the Stiftung f€urPathobiochemie undMolekulare

Diagnostik and the Roggenbuck Foundation (K. Bartkowiak), LEXI Forschungs-verbund "Surface Targeting" (T.M. Gorges, S. Riethdorf, and K. Pantel), and ERCAdvanced Investigator Grant DISSECT (K. Pantel).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 18, 2014; revised July 30, 2015; accepted September 2,2015; published OnlineFirst November 16, 2015.

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2015;75:5367-5377. Published OnlineFirst November 16, 2015.Cancer Res Kai Bartkowiak, Marcel Kwiatkowski, Friedrich Buck, et al. Protein ResponseCancer Patients through Sustained Activation of the Unfolded Disseminated Tumor Cells Persist in the Bone Marrow of Breast

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